Fig. 6. RhoA effectors ROCK and MLCK mediate assembly of rolipram-sensitive adhesion microspikes. (A) Cells were plated onto laminin under control conditions (upper panel) or in the presence of the ROCK inhibitor Y27632 (10 µM) (lower panel). Cells were fixed 1 hour after plating and stained for vinculin (green) and actin (red). (B) Cells were allowed to adhere to laminin coated plates in the absence (–) or presence (+) of rolipram. Cells were lysed 1 hour after plating and the phosphorylation state of MLC was determined by western blotting using an antibody specific for MLC phosphorylated at Ser19. Levels of total MLC were determined by using an antibody that recognises unphosphorylated and phosphorylated MLC equally well to indicate equal loading. The right panel represents the data of three independent experiments (mean±s.e.m.); phospho-MLC levels were determined by densitometry and normalised to the amount of total MLC. (C) Cells were plated onto laminin under control conditions. After 1 hour cells were fixed and stained for MLC phosphorylated at Ser19 (green) and actin (red). (D) Experimental set-up like in (C) except that rolipram (10 µM) was included. (E) Experimental set-up like in (C) except that Y27632 (10 µM) was included. Solid arrows indicate peripheral phospho-MLC staining only observed in the absence of inhibitors (C). Cytoplasmic phospho-MLC staining, observed under all conditions, is indicated by dotted arrows (C-E). Scale bars, 20 µm (A) and 40 µm (C-E).