Fig 16. Effect of MLCK knockdown or inhibition on LC₂₀ phosphorylation under serum-free conditions.

(A) MLCK was down-regulated in CASMC by siRNA treatment. Controls were treated identically, but with negative control siRNA. Cells were serum starved overnight prior to lysis in Laemmli sample buffer for Phos-tag SDS-PAGE and western blotting with anti-LC₂₀. A representative western blot (left-hand panel) and cumulative quantitative data (right-hand panel) are shown. Data are presented as LC₂₀ phosphorylation stoichiometry and values represent the mean ± SEM. “ns” denotes p > 0.05 (n = 10; Student’s unpaired t-test). (B) Effect of ML7 (10 μM) treatment on LC₂₀ phosphorylation (analysed by Phos-tag SDS-PAGE and western blotting with anti-LC₂₀) in the absence of serum stimulation. Controls (C) were incubated with vehicle rather than ML7. A representative western blot is shown in the left-hand panel and overall phosphorylation stoichiometry in the right-hand panel. “ns” denotes p > 0.05 (n = 6; Dunnett’s post hoc test). (C) Effect of wortmannin (1 μM) treatment on LC₂₀ diphosphorylation (analysed by western blotting with anti-2P-LC₂₀: representative western blots are shown in the upper panel with cumulative quantitative data in the lower panel. “ns” denotes p > 0.05 (p = 0.8633; n = 11; Student’s unpaired t-test). (D) Effect of chelation of Ca²⁺ with EGTA on LC₂₀ phosphorylation under serum-free conditions. CASMC were incubated in medium containing Ca²⁺ (2.5 mM) or EGTA (5 mM) under serum-free conditions prior to Phos-tag SDS-PAGE and western blotting with anti-LC₂₀. A representative western blot is shown in the upper panel with quantitative data depicted in the lower panel. “ns” denotes p > 0.05 (Student’s unpaired t-test).