Fig. 7. Phosphorylated wild-type L-plastin and S/E variant bind with a higher stoichiometry to F-actin than non-phosphorylated wild-type protein. (A) Binding of phosphorylated and non-phosphorylated wild-type L-plastin to F-actin. Wild-type L-plastin (L-plastin WT) was incubated in the absence or presence of the catalytic domain of PKA for 120 minutes at 30°C. G-actin (6 µM) was copolymerised with various concentrations of phosphorylated [L-plastin WT (P)] or non-phosphorylated wild-type L-plastin (L-plastin WT) and centrifuged at high speed. Supernatants and pellets were analysed by SDS-PAGE. Coomassie-staining patterns of pellets (top panel) and supernatants (bottom panel) of triplicate samples are shown. The molar ratios of L-plastin to actin are indicated. (B) Quantification of binding of phosphorylated or non-phosphorylated wild-type L-plastin and L-plastin S/A variant to F-actin. Amounts of phosphorylated (black) or non-phosphorylated (blue) wild-type L-plastin and L-plastin S/A variant (red) in pellets and supernatants were quantified by densitometry of Coomassie-stained protein bands. L-plastin in the pellets is plotted as a function of increasing L-plastin concentrations. Each point is the mean of three experiments ± s.d. (C) Immunoblotting analysis of phosphorylated wild-type L-plastin, co-sedimented with F-actin. A fraction of protein pellets shown in A (upper panel) was analysed by immunoblotting using anti-Ser5-P antibody (lower panel). After stripping the membrane, total L-plastin was detected with anti-L-plastin antibody (upper panel). Triplicate samples are shown for each actin to plastin ratio. (D) Binding of WT L-plastin and S/E variant to F-actin. G-actin (12 µM) was copolymerised with various concentrations of WT L-plastin or S/E variant and centrifuged at high speed. Coomassie-staining patterns of supernatants (S) and pellets (P) are shown. The molar ratios of L-plastin to actin are indicated. (E) Binding curves of WT L-plastin or S/E variant to F-actin. Amounts of F-actin-bound WT L-plastin (rhombus) and S/E variant (square) in the pellet are plotted as a function of increasing L-plastin concentrations. Each point is the mean of four experiments ± s.d.