Phosphorylation of RLCs in relaxed (pCa >8) and stimulated (pCa 4.3) skinned bladder strips. Isometrically mounted strips underwent the same experimental protocol as in Fig. 8. The strips were fixed in TCA either after a full contraction/relaxation cycle (pCa >8, relaxed) or at the plateau of the subsequent maximal contraction (pCa 4.3, contracted). (A) Representative silver-stained 2-D–PAGE. MLC17, 17-kD light chain; U, unphosphorylated smooth muscle RLC (smRLC); P1, monophosphorylated nmRLC; P2, unphosphorylated nmRLC + diphosphorylated smRLC; P3, monophosphorylated smRLC. (B) Phos-Tag 2-D–PAGE followed by Western blots with antibodies against RLC and RLC phosphorylated at S19. (C and D) Summary of results of densitometry of silver-stained 2-D–PAGE. RLC isoelectric variants were expressed in percentage of total RLC (sum of P1, P2, P3, and U). Bars represent mean ± SD, and symbols are individual fetuses (relaxed: seven WT and six KO; contracted: eight WT and seven KO). *, P < 0.05; *** P < 0.001 (comparison between genotypes, two-way ANOVA; contracted versus relaxed within one group with one-way ANOVA, both followed by Sidak’s multiple comparison test).