Subramanian Aasha Research Paper

Student Information: Aasha Subramanian
Grade 12, Centennial High School

[email protected]
Mentor Information: Dr. Lakshmi Santhanam, PhD

[email protected]
Research Paper Title: Optimizing ectopic lysyl oxidase like 2 (LOXL2) expression in mammalian cells using
viral titration (Biomedical Sciences, Molecular/Cellular)
Statement of Outside Assistance: Research involving mammalian cell lines was conducted at Johns Hopkins
University Ross Research Lab under the supervision of Dr. Lakshmi Santhanam, assistant professor of
Anesthesiology and Critical Care Medicine and Biomedical Engineering at the Johns Hopkins University School of
Medicine. All the materials were provided by the research lab.
Abstract
Lysyl oxidase like 2 (LOXL2) is a member of the lysyl oxidases (LOX) superfamily of copper-dependent enzymes
that are primarily involved in the creation, maintenance, and remodeling of the extracellular matrix (ECM) in
animals through the cross-linking of collagen and elastin fibers. Studies suggest that LOXL2 has the main purpose
of matrix deposition. However, the protein may have catalytically independent functions in cells. This is suggested
by the presence of four scavenger receptor cysteine rich (SRCR) domains, along with a lysyl oxidase (LOX) domain
in the LOXL2 protein. To fully understand the scope of LOXL2’s functions in cells, several viral adenoviral vectors
to express different versions of LOXL2 were generated in my mentor’s laboratory 1) the full length LOXL2 (AdFL),
2) LOXL2 lacking the first two SRCR domains (AdΔN), 3) LOXL2 with the LOX domain deleted (AdΔLOX), and
4) full length LOXL2 with a double mutation in the LOX domain that makes the protein catalytically dead
(LOXL2-DM).
In this paper, the main goal was to determine the efficacy of these adenoviruses and optimize the concentration and
duration of expression to obtain similar levels of expression of these different LOXL2 proteins in mammalian cells.
To this end, LOXL2 (lysyl oxidase like-2) and the prototypic member of this superfamily, lysyl oxidase (LOX) were
probed under four different conditions. The conditions involved delivering two different quantities of four viruses
(AdFL, AdΔN, AdΔLOX, AdDM) to compare the protein expressions of LOX and LOXL2 among the conditions,
allowing for adjustment in future trials if there was significant inconsistency. Western blotting was used to separate
and identify proteins based on molecular weight and by type using gel electrophoresis. After analyzing the protein
expression and normalization volumes of the proteins, it was found that the quantifications of 1μL of AdFL, 5μL of
AdΔN, 2μL of AdΔLOX, and 2μL of AdDM yielded the most similar levels of LOXL2 mutant overexpression.
Using this information, it is possible to determine preliminary standards necessary to perform further experiments to
research the protein assembly and catalytic function of the LOX and LOXL2 proteins.
Table of Contents
1. Introduction
2. Materials
3. Methods and Procedures
1. Cell Culture
2. Gel Preparation
3. Gel Electrophoresis
4. Electrotransfer
5. Blocking and Antibody Incubation
4. Results
5. Discussion
6. Conclusion
7. References
Introduction
Lysyl oxidases (LOX) are a superfamily of copper-dependent enzymes that oxidize amine substrates to reactive
aldehydes. The role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals through the
cross-linking of collagen and elastin fibers (6). Amine oxidases catalyze the oxidation of primary amines (NH2) to
aldehydes (-CHO) with the release of water and hydrogen peroxide, which requires one copper ion per subunit and
topaquinone as a cofactor. [Figure 1]. The LOX family of proteins is essential for the biosynthesis and maintenance
of the ECM of cells and the stabilization of tissues in the cardiovascular system. Collagen, a tough, strong fiber, is
the most abundant protein in the body and provides tensile and compressive strength to organs and tissues. Elastin is
a stretchy and resilient protein that helps return tissues to their previous state after being stretched. Collagen and
Elastin proteins are essential for the formation of the extracellular matrix (ECM) of cells. Lysyl oxidase superfamily
contains five enzymes: LOX and LOX-like (LOXL) 1 through 4. LOXL2 is highly expressed in the blood vessels
along with the prototypical LOX. The SRCR domains present in LOXL2 are considered to be motif-recognition
domains, which help to bind other proteins. Therefore, it is proposed that LOXL2 can have a protein assembly
function through these SRCR domains, independent of its amine oxidase function.
Lysyl Oxidase-Like 2 (LOXL2) is a member of the LOX family that may elevate the risk of older people having a
heart attack or stroke if overexpressed. This happens by the ability of LOXL2 to remodel the matrix in arteries.
Aortic remodeling causes aortic stiffening, which in turn can lead to diseases such as Isolated Systolic Hypertension
(ISH), cardiac hypertrophy, heart failure, and interstitial fibrosis (16). Aortic remodeling can happen with natural
aging and be caused by other diseases like hypertension, obesity, and diabetes. In these settings, LOXL2 deposits
more collagen than elastin, which makes the aorta stiffer (due to excess collagen) and less stretchy (due to less
elastin).
Another way that LOXL2 can cause aortic stiffening is by acting as a protein assembly molecule. In this case, the
LOXL2 would bind and organize the collagen fibers, which would then allow LOX to catalyze the crosslinking.
Given that there are two possible ways in which LOXL2 can cause aortic stiffening, it is important to know which of
these functions of LOXL2 is the most important contributor. Then, it would be possible to develop drugs to
specifically target that function to reduce heart attacks and strokes.
A common way in which scientists study biochemical functions of a protein is by first overexpressing the protein in
a relevant cell type. In this case, the goal is to overexpress LOXL2 in aortic smooth muscle cells, which are one of
the important cell types in the blood vessel. Moreover, because LOXL2 can have more than one function in cells,
the goal is to deliver either the intact protein or mutants of the protein that will selectively retain only one of its
functions. A common way to deliver proteins to mammalian cells is to use adenoviruses that have been engineered
to overexpress the protein of interest, but that do not self-replicate to protect the person performing the experiment.
The adenoviruses contain protein coding genes that can be expressed using viral vectors, which can label cells to
track them and their progeny, commonly to study the function of a particular protein.
Therefore, in this project, a viral titration was performed in Human Aortic Smooth Muscle Cells (HASMC) and Rat
Thoracic Aortic Smooth Muscle Cells (A7R5) cell lines in order to overexpress the LOXL2 protein. The overall
goal was to optimize the concentration of virus and duration of overexpression with the purpose of normalizing the
amount of expression. The results will help us further study the specific biology and biochemistry of LOXL2 in
cells, which can later be translated to animal models for the discovery of new ways to regulate LOXL2 expression
and activity in diseases.
Figure 1. LOX family members contain a conserved COOH-terminal domain consisting of a catalytic domain with a
copper-binding motif and a lysyl-tyrosyl-quinone (LTQ) cofactor. The amino termini of the LOX proteins are more
variable and may a role in substrate specificity and function (4).
With the goal of studying the functions of different LOXL2 domains, four different adenoviral vectors were used:
The first one was AdLOXL2 Full Length, which possesses both the LOX and SRCR domains, and represents the
native LOXL2 protein. This protein will have both the amine oxidase activity to crosslink collagen and elastin, and
the ability to assemble proteins by its SRCR domains. The second vector, N-terminal (ΔN), has the SRCR domains
1 and 2 deleted from the full length protein structure. Based on prior studies in the mentor’s laboratory, it is
predicted that the first 2 SRCR domains play a significant role in LOXL2’s function in cells. This protein has SRCR
3-4 and the LOX domain. It is predicted that this version of the protein will therefore lack the assembly function and
only retain the amine oxidase function. The third vector, ΔLOX, removes the proteolytic LOX domain of the protein
and expresses only the SRCR-domains. This domain will therefore enable us to analyze the function of the SRCR
domains in isolation. The fourth vector is a double mutant (DM), in which two amino acids crucial for its enzymatic
activity were mutated out, rendering the expressed protein catalytically inactive. This protein will enable us to see if
all the four SRCR domains are needed for the protein assembly function, or if SRCR 1-2 are necessary and
sufficient for this function by comparing it to the ΔN mutant.
Virus titration is performed in order to determine the amount of viral delivery needed to achieve the same protein
expression under each condition. The hypothesized amount of virus delivery necessary to yield an equal protein
expression is 0.5uL of AdFL, 1uL of AdFL, 5uL of AdΔN, 10uL of AdΔN, 5uL of AdΔLOX, 10uL of AdΔLOX,
5uL of AdDM, and 10uL of AdDM over the course of 24 hours. Before further studies can be performed to expand
our knowledge on the LOX protein and other members of the LOX super family, virus titration must be completed
to ensure the results are caused by the protein function and not expression level.
Materials and Methods

2.1 Virus Titration
The virus titration experiment was performed in two cell lines: HASMC and A7R5 (12). In both cell lines, four
viruses causing the overexpression of LOXL2 or its mutants were delivered to the cells: AdLOX Full Length
(AdFL), AdLOXΔN (AdΔN), AdLOXΔLOX (AdΔLOX), and AdLOXDM (AdDM). In the HASMC experiment,
cells were plated on 6-well plates. After overnight cell adhesion, cells were serum starved in DMEM/F-12 media
with ITS to achieve cell cycle arrest. Control well received no virus, and each remaining well received one of the
following conditions: 0.5uL of AdFL, 1uL of AdFL, 5uL of AdΔN, 10uL of AdΔN, 5uL of AdΔLOX, 10uL of
AdΔLOX, 5uL of AdDM, and 10uL of AdDM. Cell media was collected after 24 hours of virus delivery, and cells
are washed and scraped in lysis buffer for western blotting.
To further normalize the quantity of protein expression to produce an equal expression, the experiment was repeated
in the A7R5 cell line. The viruses remained the same, however the cells were divided into two sets, one ran for 24
hours and the other for 48 hours. Different quantities of virus were delivered to the cells: 1ul of AdFL, 2ul of AdFL,
2ul of AdDM, 5ul of AdDM, 2ul of AdΔN, 5ul of AdΔN, 2ul of AdΔLOX, 5ul of AdΔLOX. It is expected to detect
proteins including collagen, elastin, FN (fibronectin), in the extracellular matrix (ECM) of the cell, as these are
structural proteins of the ECM. We examined LOX, LOXL2 and LOXL3 levels in the extracellular matrix as they
are the relevant forms of lysyl oxidases in the smooth muscle cells. The purpose of this experiment was to observe
the relationship between the amount of virus delivered to cells and time the experiment is conducted on the amount
of protein expression.
2.2 Cell Culture/ Passaging:

In preparing for the cell culturing, all materials were ensured to be sterile and functioning before the experiment was
conducted.
Procedure used to passage cells is as follows. The existing media was suctioned off of the 100mm plate and washed
once with 1 mL PBS. In order to detach the cells from the plate, 1.5mL of trypsin was delivered and incubated for
three to five minutes. The cells were checked under the microscope to confirm that they were detached. 1.5 mL of
complete media was measured and added to neutralize the cells (11). The cells and solution were resuspended
evenly throughout the plate by pipetting the cells to break up the clumps, ensuring that it is mixed thoroughly. The
cell suspension was collected and split equally into a 6-well plate. [Figure 2]. An appropriate amount of complete
media was added to get a total of 2 mL inside each well. The wells were gently rocked back and forth as well as side
to side to help distribute the solution in the wells.
Figure 2. Shows subculture procedure (2).
In order to deliver the virus to the cells, media was first suctioned from the wells and then 1.5mL of fresh media
with 1% ITS (Insulin-transferrin- selenium) was added into each well. The appropriate calculated amount of virus
was delivered to the cells.
2.3 Gel Preparation:

To prepare cells for running a western blot from the six well plate, the ITS media was first collected into centrifuge
tubes. Phosphate buffered saline (PBS) was added to rinse the wells to remove any remaining media. This step was
repeated once more. Afterwards, 20uL of 3X loading buffer was pipetted into the wells. Each well was scraped with
a sterile cell scraper and collected into a centrifuge tube. An extra 10uL of 1.5X loading buffer was added to each
pellet sample. To concentrate protein in media samples, 10 uL of StrataClean resin was added into each media
sample. To maximize the interactions between resin and media protein, the solution was vortexed at medium speed
for 5 minutes. The media samples were then centrifuged at 10,000 rpm for five minutes. The supernatant was
suctioned off and the pellet was resuspended in 25 uL of 1.5X loading buffer. The pellet and media samples were
boiled at 100 degrees Celsius for 15 minutes, cooled on ice for one minute, and centrifuged for one minute at 10,000
rpm to prepare the samples to be loaded into the gel (2).
2.4 Gel Electrophoresis:

For western blotting, we used Bio-Rad premade mini or midi gels. In the electroporator, 1X running buffer was
poured to above the well openings. (7). The first well was loaded with 4uL of protein standard. The other wells
were loaded with the protein samples. Media and pellet samples were run on separate gels. The gel was run at 200
volts for 30 minutes for mini gels or 45 minutes for midi gels until the dye sinks to the bottom of the gel [Figure 3].
Figure 3. Illustration of protein electrophoresis equipment utilised in separating protein fragments by size and
molecular weight. The samples are placed in wells at one end of the gel and an electrical current passes through the
gel, allowing the smaller proteins to move towards the positive electrode faster than the larger proteins (15).
2.5 Electrotransfer:
Filter sheets and nitrocellulose paper were cut to fit the measurement of the gel to prepare for the protein transfer.
The gel plate was placed into water and separated using a splitting key, and the gel is flipped onto a piece of
nitrocellulose paper. Sandwiched by filter papers, the gel and nitrocellulose paper were placed in the electrotransfer
tray. To ensure that there are no air bubbles between the gel and nitrocellulose paper or the filter papers, a rolling
tool was used to gently push over the surface to release the gaps between the materials [Figure 4]. The sandwich is
placed in a transfer tray and inserted into the Bio-Rad Turbo Transfer machine. The transfer was able to occur as the
negatively charged proteins on the gel attracted to the positively charged nitrocellulose paper, creating an electrical
current on the concentration gradient and a great amount of pressure, allowing the proteins to move onto the
nitrocellulose paper (9).
Figure 4. Typical semi dry transfer setup in electrotransfer tray.

2.6 Western Blotting (Blocking and Antibody Incubation):
After transferring the proteins from the gel to the nitrocellulose membrane, the blot was immersed into Ponceau S
Staining Solution in a container and stained for five minutes on a shaker. The blot was placed between two clear
plastic sheets and into the chemiluminescent machine to take a picture of the ponceau staining using the colorimetric
setting. This allows visualization for the lanes of protein before probing for a specific protein. The western blot was
washed for approximately five minutes on the shaker with TBST (Tris Buffered Saline with Tween), a solution
containing pH stabilizers, salts and detergents designed to effectively remove excess material from membranes
without disrupting the antibody binding reaction. Unbound materials were washed away without suppressing
antigen-antibody binding interactions, thereby reducing nonspecific background and increasing the specific signal.
After wash step, 3% milk was added. The milk is used to block the nonspecific binding sites so that the antibody can
attach to its specific binding site (7). The blocking agent was shaking in the container with the western blot for about
one hour. The blot was rinsed with TBST three times for five minutes each time, and the primary antibody was
added to the blot in 3% milk and shook at low speed for at least 1 hour. This antibody specifically binds to the
protein of interest (1). The blot was washed with TBST in the same amount of repetitions and time between each
step of antibody binding when probing for a protein. The appropriate secondary antibody was added to bind onto the
primary antibody in order to detect the protein and produce an amplification in signal [Figure 5]. Rabbit primary
and Anti-Rabbit secondary were used to detect LOX and LOXL2 proteins. The primary antibody was left from
anywhere between an hour and overnight on the shaker, while the secondary antibody was only left for an hour to
avoid binding onto non targeted binding sites (1). To image the blots, An ECL (enhance chemiluminescent
substrate) mix was prepared following the proportion of solution A and B provided by the manufacturer. The blots
were washed, placed onto a clear film, the ECL solution was spread evenly onto the blot, and the blot was enclosed
with another plastic sheet. This sheet was placed into the chemiluminescent machine and a picture of the blot was
taken to detect the protein expression in each lane (3). The ECL produced a chemical reaction that was detected by
the chemiluminescent machinery, allowing for protein recognition. In order to probe for a different protein, stripping
buffer was added to the western blot and placed on the shaker for approximately 10 minutes to strip the previous
primary and secondary antibodies. Once this was finished, the process was repeated from the blocking to imaging
step with the new antibodies on the same blot.
Figure 5. The antibody incubation process of western blot after protein transfer onto nitrocellulose paper.
Results (Data or Findings)

In order to analyze the western blots, a densitometry analysis was performed to select and determine the
background-subtracted density of the bands using Band Analysis tools in the ImageLab software. The analysis
revealed the amount of expression of the specifically probed proteins (LOX, LOXL2, FN, or LOXL3) in each lane
and band of the blots compared to the standard Ponceau S Staining, which contained the normalized protein lanes.
In comparing the protein expression of LOX produced by the overexpression of LOXL2 mutants amongst the A7R5
cell samples tested in the media and pellet, the results revealed similar amounts of expression over the 24 hour
duration compared to the 48 hour period. The protein expression in 1uL of AdFL, 5uL of AdΔN, 2uL of AdΔLOX,
and 2uL of AdDM in the media was relatively similar in spite of the outliers which were the control, 2uL of AdFL,
and 5uL of AdDM [Figure 6]. However, the protein expression in the same experiment tested after 48 hours yielded
a great range of variation in expression [Figure 8]. The results generated from the pellet samples were similar to that
of the media, as the protein expression for 1uL of AdFL, 5uL of AdΔN, 2uL of AdΔLOX, and 2uL of AdDM,
contained a similar amount of expression in the 24 hour time period [Figure 7], while expression varied in the
results when the same values were measured after 48 hours [Figure 9].
The LOX expression in the HASMC cells in media was low and variable with no particular trend with the addition
of adenoviruses [Figure 10]. The LOXL3 expression in the pellet was similar amongst the four conditions: 1uL of
AdFL, 5uL of AdΔN, 5uL of AdΔLOX, and 5uL of AdDM [Figure 11]; the protein expression from 10uL of
AdDM was an outlier as it yielded a much greater expression compared to the other viruses.
Western Blot; Anti-LOX in A7R5 cells expressing ADLOXL2 Mutants (Media)
Figure 6. The graph shows the percentage of LOX protein expression in the given conditions normalized to standard
ponceau staining present in the media of A7R5 cell samples testing ADLOXL2 Mutant viruses after 24 hours.
Western Blot; Anti-LOX in A7R5 cells expressing ADLOXL2 Mutants (Pellet)
ponceau staining present in the pellet of A7R5 cell samples testing ADLOXL2 Mutant viruses after 24 hours.
Western Blot; Anti-LOX in A7R5 cells expressing ADLOXL2 Mutants (Media)
Western Blot; Anti-LOX in A7R5 cells expressing ADLOXL2 Mutants (Pellet)
Western Blot; Anti-LOX in HASMC cells expressing ADLOXL2 Mutants (Media)
Figure 10. The graph shows the percentage of LOX protein expression in the given conditions normalized to
standard ponceau staining present in the media of HASMC cell samples testing ADLOXL2 Mutant viruses after 24
hours.
Western Blot; Anti-LOXL3 in HASMC cells expressing ADLOXL2 Mutants (Pellet)
Figure 11. The graph shows the percentage of LOXL3 protein expression in the given conditions normalized to
hours.
In comparing the LOXL2 expression produced by the overexpression of LOXL2 mutants in the A7R5 cell samples,
1uL of AdFL, 5uL of AdΔN, 2uL of AdΔLOX, and 2uL of AdDM produced a relatively close protein expression
when the media was tested after 24 hours in the samples [Figure 12]. Similar results were found in the pellet after
the 24 hour duration [Figure 13], as the protein expression in the 1uL of AdFL, 5uL of AdΔN, 2uL of AdΔLOX,
and 2uL of AdDM were similar. The 48 hour experiment yielded very different results in the media and pellet
[Figures 14 and 15] because half of the samples contained little to no protein expression while 1uL of AdFL, 2uL
of AdFL, 2uL of AdΔN, and 5uL of AdΔN produced protein expression. The bands that contained protein
expression however, varied drastically as well.
HASMC cells with addition of adenoviruses for 48 hours yielded potent overexpression for all LOXL2 mutants
dose-dependently. Addition of AdLOXL2 1ul, AdLOXL2 dN 10ul, AdLOXL2 dLOX 10ul and AdLOXL2 DM 5ul
produced similar levels of LOXL2 mutant in media secretion. [Figure 16]. The LOXL2 expression in the pellet was
similar across the conditions: 0.5uL of AdFL, 1 uL of AdFL, 10uL of AdΔN [Figure 17]. There was a large range of
protein expression in the pellet as there was in the media.
Western Blot; Anti-LOXL2 in A7R5 cells expressing ADLOXL2 Mutants (Media)
standard ponceau staining present in the media of A7R5 cell samples testing ADLOXL2 Mutant viruses after 24
hours.
Western Blot; Anti-LOXL2 in A7R5 cells expressing ADLOXL2 Mutants (Pellet)
standard ponceau staining present in the pellet of A7R5 cell samples testing ADLOXL2 Mutant viruses after 24
hours.
Western Blot; Anti-LOXL2 in A7R5 cells expressing ADLOXL2 Mutants (Media)
Red – indicating powerful signals that caused over-exposure of WB.
standard ponceau staining present in the media of A7R5 cell samples testing ADLOXL2 Mutant viruses after 48
hours.
Western Blot; Anti-LOXL2 in A7R5 cells expressing ADLOXL2 Mutants (Pellet)
standard ponceau staining present in the pellet of A7R5 cell samples testing ADLOXL2 Mutant viruses after 48
hours.
Western Blot; Anti-LOXL2 in HASMC cells expressing ADLOXL2 Mutants (Media)
hours.
Western Blot; Anti-LOXL2 in HASMC cells expressing ADLOXL2 Mutants (Pellet)
standard ponceau staining present in the pellet of HASMC cell samples testing ADLOXL2 Mutant viruses after 24
hours.
When comparing the FN protein expression in the A7R5 cell samples, the results showed that 2uL of AdΔN, 5uL of
AdΔLOX, and 2uL of AdDM produced similar protein expressions in the media over the 24 hour duration [Figure
18]. The FN protein expression in the pellet after 24 hours yielded dramatically different results because 5uL of
AdΔLOX and 2uL of AdDM were outliers that produced more than twice the protein expression of the other lanes
[Figure 19]. The protein expressions over 48 hours contained inconsistent results in the media and pellet [Figure 20
and 21] as there was a great variation in expression among the conditions. The protein expression of FN in the
HASMC media cell samples were similar among the conditions half the conditions while the others (0.5uL of AdFL,
5uL of AdΔN, 10uL of AdΔN) produced drastically less expression. The results were very different in pellet
samples as 1uL of AdFL had a greater protein expression compared to the other conditions, which produced little to
no expression [Figure 22 and 23].
Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Media)
Figure 18. The graph shows the percentage of FN protein expression in the given conditions normalized to standard
Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Pellet)
Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Media)
Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Pellet)
Western Blot; Anti-FN in HASMC cells expressing ADLOXL2 Mutants (Media)
ponceau staining present in the media of HASMC cell samples testing ADLOXL2 Mutant viruses after 24 hours.
Western Blot; Anti-FN in HASMC cells expressing ADLOXL2 Mutants (Pellet)
ponceau staining present in the pellet of HASMC cell samples testing ADLOXL2 Mutant viruses after 24 hours.
Discussion
The results of the viral titrations were inconsistent as can be seen in the variation in the protein expressions in LOX,
LOXL2, LOXL3, and FN. The inconsistency may be a result of minimal replications, as this experiment only
contained no repetitions for any conditions for the media and pellet: one set of AdLOXL2 mutants were tested over
a 24 hour duration and another in a 48 hour duration. This experiment must be repeated many more times in order to
produce similar results, from which we can quantify the average protein expression generated from the specific
conditions. There may have also been errors in measuring or pipetting the virus to the cells, which is a common
technique error in laboratory experimentation for beginners that affects the results drastically. For example, if a
small excess amount of the AdFL virus is added to the cell samples, there could be oversaturation in the protein
expression results since it is a more powerful virus compared to the others used [Figure 10, 12 and 23]. On the
contrary, if less virus is added to the cell samples or a specific virus is not very potent, the results may be similar to
or less than the control meaning the overexpression was not successful- this can be seen in at least one or two lanes
in most western blots (Figure 23 is one example). Cases in which the overexpression is only present in the media
but not the pellet and vice versa may be a result of error in the experiment procedure or antibody specificity [Figure
7 versus Figure 6]. For example, if a protein is overexpressed only in the media, there may be human error where
cells weren’t properly scraped for sample preparation, or the LOXL2 mutants might lack specific domains to stick to
the extracellular matrix. Another reason for the overexpression to only be detected in the scraped cell portion, is that
important domains might be absent in the mutant construct and prevent transport to the extracellular matrix, trapping
the mutant proteins intracellularly. The protein expression of FN (fibronectin) indicates the level of crosslinking of
the extracellular matrix proteins among the conditions with different LOXL2 mutant overexpression. There is often
a positive correlation between FN accumulation and LOXL2 activity meaning that more FN protein deposited in the
extracellular matrix may have resulted from higher LOXL2 protein expression and activity. Further experimentation
and more repetitions will be required to gain a better understanding of the amount of ADLOXL2 mutant virus
necessary to yield similar protein expressions under the specific conditions presented. After analyzing the data, the
original hypothesis was not supported, as the results revealed that the predicted amounts of virus delivery produced
very inconsistent protein expressions that had to be adjusted to meet the goal. The virus adjustment revealed that
1uL of AdFL, 5uL of AdΔN, 2uL of AdΔLOX, and 2uL of AdDM amounts of virus delivery tested after 24 hours in
the A7R5 cell samples produced the most similar results in the media and pellet when probed for LOX and LOXL2
with the given results. More experiments need to be conducted to determine the whether FN deposition is increased
with the addition of AdLOXL2 mutant virus and LOXL2 overexpression.
Conclusion
If more trials of this experiment are tested in the future, it would be favorable to adjust the amount of virus added to
a slightly lower quantity. The suggested increment of virus addition will be 0.5uL lower for each condition, in order
to further normalize the protein expression. The results showed that lowering the quantities of virus over the 24 hour
course of growth allowed the purpose of the experiment to be better fulfilled.
Human errors involving consistency in performing the methods and procedures may have skewed the results of the
experiment. Executing more trials of the same quantities and adjusting the amount of virus to the suggested lower
quantities will be beneficial in analyzing the most optimal conditions (amount of virus to be delivered and
experiment duration) to produce a similar amount of protein expression. This simple experiment is important in
researching the functions of the SRCR domains, which forms the foundation of a larger assay to analyze the
functions of different LOXL2 domains, whether it be protein assembly or catalytic function. Studying the function
and activity of LOX, LOXL2, LOXL3 with biochemistry experiments involving these mutant virus will allow
scientists to better characterize these amine oxidases that can play a role in pathogenesis of cardiovascular diseases.
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Subramanian Aasha Research Paper

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Student Information: ​Aasha Subramanian

Grade 12, Centennial High School

Mentor Information:​ Dr. Lakshmi Santhanam, PhD

Materials and Methods

2.2 Cell Culture/ Passaging:

Figure 2. ​Shows subculture procedure (2).

2.3 Gel Preparation:

2.4 Gel Electrophoresis:

Figure 4. ​Typical semi dry transfer setup in electrotransfer tray.

Results (Data or Findings)

Western Blot; Anti-LOX in A7R5 cells expressing ADLOXL2 Mutants (Media)

Western Blot; Anti-LOX in A7R5 cells expressing ADLOXL2 Mutants (Media)

Western Blot; Anti-LOX in HASMC cells expressing ADLOXL2 Mutants (Media)

Western Blot; Anti-LOXL2 in A7R5 cells expressing ADLOXL2 Mutants (Pellet)

Western Blot; Anti-LOXL2 in A7R5 cells expressing ADLOXL2 Mutants (Pellet)

Western Blot; Anti-LOXL2 in HASMC cells expressing ADLOXL2 Mutants (Pellet)

Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Media)

Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Pellet)

Western Blot; Anti-FN in A7R5 cells expressing ADLOXL2 Mutants (Pellet)

Western Blot; Anti-FN in HASMC cells expressing ADLOXL2 Mutants (Pellet)

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Student Information: Aasha Subramanian

Mentor Information: Dr. Lakshmi Santhanam, PhD

Figure 2. Shows subculture procedure (2).

Figure 4. Typical semi dry transfer setup in electrotransfer tray.