Fig. 2. Suppression of aPKC activity does not affect cell-cell-contact-induced myosin-II activation. (A) MTD1-A cells transfected with adenovirus vectors encoding β-galactosidase (LacZ) or aPKC kn were subjected to a Ca²⁺ switch as described in the legend for Fig. 1, fixed at the indicated times after the Ca²⁺ switch, and double stained with a mixture of antibodies against nonmuscle myosin heavy chain IIA and IIB (magenta in merged images) and against -actinin (green in merged images). Arrows, circumferential actin cables; arrowheads, perijunctional actin belts. (B) Control MTD1A cells were fixed at 30 minutes after a Ca²⁺ switch and double stained for F-actin (phalloidin; green in merged images) and with antibodies against either myosin II, Ser19 monophosphorylated myosin light-chain 2 (p-MLC2) or Thr18 and Ser19 diphosphorylated MLC2 (pp-MLC2) (magenta in merged images) as indicated at the top. Note that radial actin fibers were free from myosin II. (C) MTD1-A cells were immunostained with anti-p-MLC2 antibody at the indicated times after the Ca²⁺ switch. Pre-treatment with Y27632 completely abolishes the staining, suggesting that ROCK is dominantly responsible for this phosphorylation. (D) Total cell extracts of MTD1-A cells were prepared at the indicated times after the Ca²⁺ switch, and the changes in the p-MLC2 level were analyzed by western blotting. Note that overexpression of aPKC kn does not affect the Ca²⁺-switch-induced increase in p-MLC2. Scale bars: 10 µm.