Myosin, actin, and nonmuscle myosin heavy chain content of E18.5 bladders. (A) Representative Coomassie-stained SDS-PAGE with skeletal actin and turkey gizzard myosin as standards, and lysates from three intact WT and KO bladders each from E18.5 fetuses. Insert: calibration curve. (B–E) Summary of results from intact and skinned bladder strips of actin (Act) content (B), myosin (Myo) content (C) relative to total protein (Prot_t), molar ratio of actin to myosin (D), and actin to TM (E), the molar ratio of actin:myosin in skinned KO is significantly higher than in WT. P < 0.001; symbols represent fetuses. (F) Representative Western blot probed with antibodies against nonmuscle myosin heavy chain IIA, MYPT1, and GAPDH from intact (i) and skinned (s) urinary bladders; n = 5 fetuses per genotype. In these experiments, one half of the bladder was used intact, and the other half was skinned. Immunoreactivity was detected with the Odyssey system using IR680-conjugated (red) anti-mouse and IR800-conjugated (green) anti-rabbit secondary antibodies. (G) Summary of densitometry (a.u., arbitrary units). Symbols represent the values from determination in one fetus; bars are mean ± SD; MHC, myosin heavy chain; ns, not significant. **, P < 0.01; *** P < 0.001 (two-way ANOVA, Sidak’s multiple comparisons posttest).