FIGURE 3.

Contraction and LC₂₀ phosphorylation in intact rat caudal arterial smooth muscle in response to KCl-induced depolarization in the presence of Ca²⁺. A, intact rat caudal arterial smooth muscle strips were treated with 87 mm KCl in Ca²⁺-containing H-T buffer and the contractile response was recorded. Separate tissues were harvested at the indicated times during the contraction for analysis of LC₂₀ phosphorylation by Phos-tag SDS-PAGE and Western blotting with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Numbers below the gel lanes correspond to the time points in A. MC denotes control tissue (Triton-skinned rat caudal arterial smooth muscle treated with microcystin at pCa 9 for 60 min) to identify unphosphorylated, mono-, and diphosphorylated LC₂₀ bands. C, cumulative quantitative data showing the time course of LC₂₀ phosphorylation stoichiometry; as shown in panel b, phosphorylation occurred exclusively at Ser¹⁹ under these conditions. Values represent the mean ± S.E. (n = 3).