Fig 15. Effect of ROCK1 and ZIPK knockdown and inhibition on serum-induced ZIPK phosphorylation.

(A) ROCK1 or ZIPK expression was down-regulated in CASMC by siRNA treatment. Controls were treated identically, but with negative control siRNA. Cells were serum starved overnight and treated with 5% FBS for 2 min prior to lysis in Laemmli sample buffer for Phos-tag SDS-PAGE and western blotting with anti-ZIPK. Electrophoresis was carried out in the presence of MnCl₂ or EDTA as indicated. CASMC (B) or UASMC (C) were serum starved overnight and treated with 5% FBS for 2 min in the absence (control) or presence of GSK429286A (GSK; 1 μM) or HS38 (10 μM) prior to lysis in Laemmli sample buffer for Phos-tag SDS-PAGE and western blotting with anti-ZIPK. Representative blots are shown (n = 11 in panel A; n = 7 in panel B; n = 4 in panel C; n = 3 in panel D). Statistically significant differences in LC₂₀ phosphorylation stoichiometry from control are denoted by the actual p values (Dunnett’s post hoc test; panel B).