Hsc70 antibody

Western blot was performed as described previously [20] (link). Briefly, cells were lyzed in RIPA buffer (60 mM tris-HCl, 150 mM NaCl, 2% Na-deoxycholate, 2% Triton X-10, 0.2% SDS and 15 mM EDTA) in the presence of protease inhibitors (Roche; Complete Mini, EDTA-free). Polyclonal anti-rabbit ATP7B antibody (1∶1,000) was used for detection (kind gift of I. Sandoval, Madrid, Spain). HSC70 antibody (Santa Cruz Biotechnology) staining was used as a protein loading control.

SMCs were extracted in HEPES buffer containing 1% Triton X-100 [16 (link)]. Protein was measured using Precision-Red (Cytoskeleton) and equal amounts were then subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. Blots were developed with ECL (GE Healthcare). The following antibodies were used: anti-β-tubulin (Cell Signaling #2146), anti-smooth muscle alpha-actin (Sigma #A2547), anti-VSVG (Roche #11667351001), anti-Rac1 (BD Biosciences, #610650) and anti-RhoA (Santa Cruz sc-418). To analyze MAPK phosphorylation, serum-starved GFP-controls and SMA-SMCs were stimulated with 10% FBS and harvested at the time points indicated. Cell extracts were subjected to Western blot analysis and blots probed with antibodies to recognize the phosphorylated forms of p44/42 ERK, p38 MAPK and p54/46 JNK. All antibodies were from Cell Signaling and used at the following dilutions: #9101 (1:10,000), #9215 (1:1,000) and #9251 (1:500). To control for equal loading, blots were reprobed with HSC70 antibody (Santa Cruz, sc-7298, 1:6000).