Western blot:
RPGSMCs were cultured in 12-well culture dishes until approximately 90%
confluent before being switched to serum and growth factor starved media
for 48 hours. Cells were then washed with PBS and 1X Cell Lysis Buffer
(Cell Signaling, 9803) containing: (pH 7.5) 20 mM Tris-HCl, 1 mM
Na2EDTA, 1 mM EGTA, 1% Triton, 1 mM β-glycerophosphate,
1 mM Na3VO4, 1 μg/mL leupeptin, 2.5 mM
sodium pyrophosphate, and additional 1X protease (MilliporeSigma P8340)
and phosphatase inhibitors (MilliporeSigma, P5726) at 4ºC. A
bicinchoninic acid kit (ThermoFisher, 23225) was used to quantify lysate
protein concentration and approximately 15 μg of protein was used for
each western blot lane. Lysates were boiled at 100ºC for 10 minutes and
Laemmli buffer was added such that final lysates contained: (pH 6.8)
31.5 mM Tris-HCl, 10% glycerol, 1% SDS, 2.5% β-mercaptoethanol and
0.005% Bromophenol Blue before being loaded onto 4-12% gradient
BisTris polyacrylamide gels (Invitrogen Life Technologies, NP0335BOX).
Proteins were transferred from polyacrylamide gels to nitrocellulose
membranes (LiCor, 926-31092) and blocked for approximately 30 minutes at
room temperature with 1% BSA in PBS. Membranes were incubated in
primary antibody (Table 1) solution containing 1% BSA in PBST overnight
at 4ºC. An Odyssey CLx Imager (LiCor, 9140) was used for fluorescence
visualization and semi-quantitative analysis was performed using Image
Studio software.