Analysis of Protein Expression by Western Blotting

Roisin Quigley 16303453

This sequence of experiments was designed to test the hypothesis 'TGFbeta induces EMT and fibroblast
to myofibroblast transition (FMT) to contribute to fibrotic diseases such as pulmonary fibrosis'. A number
of experiments were designed to specifically determine the ability of TGFbeta (TGFb) to induce EMT in
lung epithelial cells (A549), and to induce FMT in ventricular human cardiac fibroblasts (VHCF). Alpha
smooth muscle actin (ASMA) protein expression in A549 and VHCF cells is known to be indicative of
EMT and FMT (Doerner and Zuraw, 2009) and is a useful parameter for the measurement of these
transitions; therefore, the aim of this experiment was to determine the effect TGF on ASMA protein
expression of these cell lines. Below are figures of data obtained when performing a Western Blot on
A549 and VHCF cells both treated and untreated with 5 ng/ml TGFb for 72 hours. Two separate gels
were loaded with protein lysates from our cell lines after TGFb treatment. Gel 1 was used for Western
Blot and Ponceau S, gel 2 was used for Simply Blue stain.

Results include a standard curve from BSA assay obtained in order to quantify ASMA protein
concentrations in our cells of interest. Results also include Simply Blue stained gel and Ponceau S stained
membrane obtained to visualize the relative protein concentrations between the samples, to ensure equal
loading of protein lysates. This will normalise the signal intensity to total protein loading. Finally
presented is the data obtained from the Western Blot for ASMA, which was performed to determine
whether TGFb induced, reduced or had no effect of ASMA expression in these cells.

Results
Bovine Serum Albumin (BSA) Concentration Standard Curve

Figure 1: Standard curve of bovine serum albumin concentrations and relative absorbance values. BSA
was diluted with RIPA buffer to known concentrations shown with a working range of 20-2,000 ug/ml.
Absorbance of all standards was read by microplate spectrophotometer @562 nm against a blank of 80ul RIPA
with no BSA. Standards and unknowns were assayed in duplicate. Equation of the line: Y= 2259.8x - 377.86.
R2 = 0.9664.

BSA standard curve with absorbance values steadily increasing with concentration. Curve is not
completely linear, a slight decrease in slope can be seen between concentrations 1500 ug/ml and 2000
ug/ml. By substituting the average absorbance values of the duplicates of unknowns for Y in the equation
of the line, protein concentrations were determined to be 1202 ug/ml (VHCF + TGF), 1273.5 ug/ml
(VHCF), 1333.5 ug/ml (A549 + TGF), 668.5 ug/ml (A549).

Simply Blue Stain

Figure 2: 10% Poly-acrylamide gel 2 run with 4 protein samples and stained with Simply Blue stain. Lane 1
(far left) contains 8 ul protein ladder for reference. Lane 2 contains protein lysate from A549 cells untreated
with TGFb. Lane 3 contains protein lysate from A549s treated with TGFb. Lane 4 contains protein from
VHCF cells TGFb treated, and lane 5 contains that of VHCF cells TGFb untreated. Protein samples were
individually diluted with laemmli buffer 4X to ensure that equal amounts of protein were loaded. 10ul protein
sample was added to each lane in a 1 ug protein to 3 ul laemmli buffer 4X ratio. Therefore, all lanes should
contain 3.3 ug protein. Samples were heated at 95°C for 5 minutes to denature proteins. Samples were then
loaded onto 10% polyacrylamide gel and run at 120 V for approx. 70minutes.

Simply Blue stain performed in order to confirm equal loading of lanes in gel. Protein ladder was stained
successfully. All sample lanes look to give an equal intensity of colour, with lane 2 possibly having a very
small decrease in intensity.

Ponceau S stain
Figure 3: Protein bands on nitrocellulose membrane stained with Ponceau S stain. After 10%
polyacrylamide gel 1 was run, protein bands were transferred to the nitrocellulose membrane. The membrane
was covered in Ponceau solution for 30 seconds then washed with water. Lane 1 contains 8 ul molecular
weight ladder for reference. Lanes 2 and 3 contain 10 ul protein sample from A549s TGF untreated and treated
respectively. Lanes 4 and 5 contain 10 ul protein sample VHCF untreated and treated respectively. Lane 6 is a
space with no protein loaded. Lanes 7-10 are arranged similarly to lanes 2-5 using 20 ul of sample.

Ponceau S stain performed to be sure that the proteins transferred from the gel onto the membrane and to
determine whether total protein loading was equal. MW ladder had strong clear intensity and definite
evidence of protein transfer can be seen. Looking at lanes 2-5 which contained 10ul cell lysate solution.
Lane 2 (A549 - TGF) has most intensity. Lane 3 (A549 + TGF) has very little intensity, but has a faint
band difficult to notice in this photo. Lanes 4 and 5 (VHCF -/+ TGF) both have equal intensity of
staining. No protein ladder was seen in lane 6 which contained no cell lysate. Looking at lanes 7-10
which contained double the protein concentration of lanes 2-5, a stronger signal was seen from all 4 lanes.
Lanes 7 and 9 (A549 – TGF and Fib - TGF) have the strongest intensity while lanes 8 and 10 (A549 +
TGF and VHCF + TGF) have slightly less intensity of colour, with lane 10 having the lowest.
Western Blot

Figure 4: Western blot analysis of protein expression in A549 and VHCF cells after exposure to X-ray film
for 10 seconds. Protein lysates were run through 10% polyacrylamide gel for 70 minutes and transferred to
nitrocellulose membrane. Membrane was blocked with 5% skim milk in TBST for 1 hr at room temperature.
Primary antibody for α-smooth muscle actin (Code A2547) was added at requisite dilution 1:7500 in blocking
solution and incubated overnight at 4°C. After this, secondary antibody, goat α-smooth muscle actin, which
was conjugated to HRP enzyme and in dilution 1:2000 in blocking solution was incubated at room temperature
for 1 hr. Membrane was then incubated with enhanced chemi-luminescent solution before being exposed to X-
ray film for 10 seconds. Lane 1: 8ul MW ladder. Lane 2: 10ul A549 – TGF. Lane 3: 10ul A549 + TGF. Lane
4: 10ul VHCF – TGF. Lane 5: 10ul VHCF + TGF. Lane 6: space. Lane 7: 20ul A549 – TGF. Lane 8: 20ul
A549 + TGF. Lane 9: 20ul VHCF – TGF. Lane 10: 20ul VHCF + TGF.

Western Blot

Figure 5: Western blot analysis of protein expression in A549 and VHCF cells after exposure to X-ray film
for 1 minute. Protein lysates were run through 10% polyacrylamide gel for 70 minutes and transferred to
nitrocellulose membrane. Membrane was blocked with 5% skin milk in TBST for 1 hr at room temperature.
Primary antibody α-smooth muscle actin (Code A2547) was added at requisite dilution 1:7500 in blocking
solution and incubated overnight at 4°C. Secondary antibody, goat α-smooth muscle actin, which was
conjugated to HRP enzyme and in dilution 1:2000 in blocking solution was incubated at room temperature for
1 hr. Membrane was then incubated with enhanced chemi-luminescent solution before being exposed to X-ray
film for 1 minute. Lane 1: 8ul MW ladder. Lane 2: 10ul A549 – TGF. Lane 3: 10ul A549 + TGF. Lane 4: 10ul
VHCF – TGF. Lane 5: 10ul VHCF + TGF. Lane 6: space. Lane 7: 20ul A549 – TGF. Lane 8: 20ul A549 +
TGF. Lane 9: 20ul VHCF – TGF. Lane 10: 20ul VHCF + TGF.

Western blot was performed to determine the effect of TGF on ASMA expression in A549 and VHCF
cells. Both the short and longer exposures yielded similar outputs. In both figures 3 and 4, 4 bands can be
seen. 2 were obtained from 10ul VHCF cell lysate samples with and without TGF treatment. Another 2
were obtained from 20ul VHCF cell lysate samples with and without TGF treatment. No bands were
obtained for any A549 cell lysate samples. Bands appear to be approx. 46 kD, consistent with the size of
ASMA.

Discussion
Figure 1: BSA standard curve

The absorbance at 562 nm for several known concentrations of BSA was measured and plotted against
concentration. The absorbance obtained increased steadily as was expected with increasing protein
concentrations. The curve is not completely linear, mainly due to the similarity in absorbance for the
highest concentrations of BSA, which could be due to a saturation in absorbance at this wavelength and
concentration. This event would affect the linear equation obtained and used to calculate protein
concentrations, therefore could be affect the final result of the western blot. Lowering the concentration
range slightly could be a way to get more accurate results. Despite this, the curve can be considered quite
accurate due to an R2 value of 0.9664; therefore, protein concentrations can also be considered relatively
accurate.

Figure 2: Simply Blue stain

The simply blue stain was successful in indicating possible pipetting errors when loading gels. Intensity
of stain remained approximately equal across all lanes, with lane 2 having a possible but very slight
decrease in intensity. All lanes should have the exact same concentration of protein, so any decrease in
intensity would indicate pipetting error. From this stain, we can conclude that pipetting was mostly
accurate.
Figure 3: Ponceau S stain

The ponceau S stain confirmed successful protein transfer from the polyacrylamide gel to the
nitrocellulose membrane. In general, the intensity of the first 4 protein loaded lanes was much less than
that of the last 4, which was expected due to the last 4 having double the amount of protein sample of the
first 4. Colour intensity between the first 4 lanes should be very similar if loaded accurately; however, in
this sample, intensity varied moderately, which suggests substantial pipetting error or other type of error
in these lanes. Ponceau S stain is useful but is known to be of low sensitivity when staining membranes
(Goldman et al., 2016). To make this experiment more accurate, pipetting errors would need to be
corrected and more sensitive stains could be considered. Variation in protein concentrations between
lanes for the Western blot may strongly discredit final results.

Figure 4 and 5: Western Blot

Results from the western blot after both short (10 sec) and long (1 min) exposure to X-ray film yielded
only bands from the VHCF lanes. Bands in VHCF lanes were expected because ASMA is an actin
isoform of the vascular smooth muscle cell type constiutively expressed both in the presence and absense
of TGFb (Zhang et al., 2009). However, It was hypothesized that TGFb would induce ASMA expression
in A549 cells, but no bands at all were obtained for any A549 cells. This was unexpected because it has
been shown in the literature that TGF is an inducer of ASMA and therefore does increase its expression in
A549 cells (Kasai et al., 2005). We expected to see a small band in the A549 cells treated with TGFb,
which would show that TGFb induces EMT with ASMA as a marker. Reasons for absolutely no bands
being seen here are unknown, although a number of human errors did occur during the experiment such
as the BSA curve being slightly off and pipetting error. Having said this, no student in the practical class
obtained any bands for A549 TGFb treated, therefore the problem was most likely common between us
all. There could have been a problem with the TGFb used to induce ASMA expression, such as treatment
not being of enough concentration or for long enough or in the methodology used. Although, when
looking through the literature many experiments that successfully show TGFb induction of ASMA
expression in epithelial cells used very similar treatment strategies (Fang et al., 2017, Zhang et al., 2009).
Another possible reason could be that the level of ASMA expression induced by TGFb in endothelial is
too low to be picked up by X-ray. This scenario should not be overlooked because even minute changes
in cell protein concentrations can have detrimental effects on its physiology. Nevertheless, from these
results it cannot be concluded that TGFb induces EMT and FMT.

References
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Zhang, M., Zhang, Z., Pan, H., Wang, D., Deng, Z. and Ye, X. (2009). TGF-β1 Induces Human
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Kasai, H., Allen, J., Mason, R., Kamimura, T. and Zhang, Z. (2005). TGF-β1 induces human
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