Anti myh

The protein levels of Rac1, MYH, SM22α, Akt, p-Akt (308) and p-Akt (473) in VSMCs were analysed by Western blot. Cells were lysed in RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China), and protein was quantified using BCA protein assay reagent (Beyotime). Proteins (25 µg) were resolved on 6% or 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes, which were blocked with 5% BSA. The primary antibodies anti-Rac1/2/3(1:1000), anti-p-Akt (308; 1:1000), anti-p-Akt (473; 1:1000) (all from Cell Signaling Technology, USA), anti-MYH (1:500), anti-SM22α (1:1000) and anti-TRPC1 (1:1000) (all from Santa Cruz, USA) were incubated with polyvinylidene fluoride membranes overnight at 4°C. After successive washes, membranes were incubated for 1 h with HRP-conjugated sheep anti-rabbit IgG (1:1000) and horse anti-mouse IgG (1:1000) at room temperature (Zhongshan Golden Bridge Biotechnology Company, China). Enhanced chemiluminescence developing methods (Beyotime) were used to visualize immunolabelling. Image-Pro Plus was used to take measurements of band density.