Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells
Fig 1
Time courses of serum-induced LC20 phosphorylation in human arterial smooth muscle cells.
Human coronary (A, B) and umbilical arterial smooth muscle cells (C, D) were seeded in 4-well plates, serum starved overnight and stimulated with 5% FBS at time zero. Cells were lysed in Laemmli sample buffer at the indicated times of serum stimulation and subjected to Phos-tag SDS-PAGE and western blotting with anti-LC20 to detect unphosphorylated (0P), mono- (1P) and diphosphorylated LC20 (2P). Representative western blots are shown in panels A and C above cumulative quantitative data for each of the LC20 bands. Panels B and D show the time courses of total phosphorylation stoichiometry, calculated as follows: mol Pi/mol LC20 = (1P + 2x2P) / (0P + 1P + 2P). Values indicate the mean ± SEM (n = 7 for panels A and B; n = 6 for panels C and D). Significant differences from the value at time zero are indicated: *p < 0.0001, †p = 0.0001 (B), *p < 0.0001, †p = 0.0023, #p = 0.0004 (D) (Dunnett’s post hoc test).