2.3. Protein extraction, PDGFRA stimulation and Western blotting

Cells were lysed in lysis buffer (20 mM Tris pH8, 50 mM NaCl, 1% NP40, complete^TM EDTA‐free Protease Inhibitor Cocktail (Roche), 2 mM activated orthovanadate (Sigma‐Aldrich) and PhosStop [Roche]). For PDGFRA stimulation, SMCs (25,000 cells/cm²) were plated on collagen I‐coated dishes in DMEM containing 10% FBS. After 24 hours, cells were rinsed twice with PBS and incubated in OPTIMEM medium without serum for 1 hour, followed by incubation with 10 ng/ml or 100 ng/ml recombinant human PDGF‐AA (Peprotech) at 37°C for 15 minutes. Cells were scraped in PBS/2 mM activated sodium orthovanadate and proteins were then extracted as before and analysed by Western blots. 32 Protein concentration was determined using the RC DCTM Protein Assay kit (BioRad). Diluted protein samples were boiled in SDS‐PAGE buffer, separated by SDS‐PAGE in 12% acrylamide/BisAcrylamide gels and transferred to nitrocellulose membranes at 100 V for 1.5 hours. Membranes were incubated according to the Odyssey technology protocol (LI‐COR Biosystems) with mouse anti‐αSMA (1:500 dilution; Santa Cruz Biotechnologies, clone 1A4), rabbit anti‐PDGFRA (1:400 dilution; Cell Signaling Technologies, #3164), mouse anti‐GAPDH (1:10 000 dilution; Sigma‐Aldrich), mouse anti‐p44/42 MAPK (Erk1/Erk2) (1:400 dilution; Cell Signaling Technologies, L34F12, #4696) and rabbit anti‐Phospho‐p44/42 (Erk1/Erk2) (Thr202/Tyr2014) (1:400 dilution; Cell Signaling Technologies, 20G11, #4696) antibodies. Immunoblots were quantified using infrared‐labelled secondary antibodies and the Odyssey infrared imaging system (LI‐COR Biosystems).