Western Blot Analysis of Smooth Muscle Cells

Cultured SMCs were washed with ice cold PBS twice, and 80 μl Laemmli sample buffer (60 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS) was added. After lysis and protein determination using a detergent-compatible protein assay from Bio-Rad (500–0116), bromophenol blue (0.01%) and β-mercaptoethanol (5%) were added to the remainder of the lysates. ≈20 μg protein was loaded per lane on Bio-Rad TGX Criterion gels and proteins were separated using Bio-Rad Tris/Glycine/SDS electrophoresis buffer at 200 V. Following separation, proteins were transferred to nitrocellulose (0.2 μm) using the Trans-Blot Turbo Transfer System (Bio-Rad) and western blotting was done essentially as described^{57 (link)}. Membranes were cut horizontally to allow for detection of multiple targets as needed, and hence blots covering the entire range of molecular weights are not available throughout. Full blots for all display items are available in the Supplementary Information file. The following primary antibodies were used: NEXN (Abcam, ab213628), P-YAP (Cell Signaling Technology,#4911), T-YAP (Cell Signaling Technology, #4912), HSP90 (BD Transduction Laboratories, 610418), calponin/CNN1 (Abcam, ab46794), SM22/TAGLN (Abcam, ab14106). Secondary anti-mouse and anti-rabbit antibodies were from Cell Signaling Technology (#7076 S, #7074 S).