Western blot analysis of regulatory proteins in Cald1^+/+ (WT) and Cald1^−/− (KO) bladders from E18.5 animals. (A) Representative Western blots: proteins of lysates were separated by 4–20% SDS-PAGE and transferred to nitrocellulose. The membranes were sequentially probed with the respective primary antibodies; immunoreactivity was visualized as in Fig. 6. Both SM22 (Western blot) and actin (Ponceau red stain of nitrocellulose membrane) were used as loading controls. (B) Summary of results of five to six determinations (one per fetus). Note different scales of y axis; there were no significant differences for the respective proteins between genotypes. (C) Expression of CPI-17 in intact and skinned fibers and summary of results; i, intact; s, skinned; CPI-17 diffuses out of skinned fibers. (D) Representative Western blots with phosphor-specific antibodies against phosphorylation of MYPT1 at T696 (pT696-MYPT1, upper panels) and T853 (pT863-MYPT1, lower panels), and antibodies against MYPT1 total in intact and skinned bladder strips; GAPDH and SM22 were used as loading controls. M, molecular weight marker proteins. (E and F) Summary of results; symbols represent individual fetuses (five KO and five WT fetuses from a total of five litters; bladders were split in half; one was homogenized intact, and the other after skinning). Bars represent means ± SD; n.s., not significant. ***, P < 0.001.