Fig. 1. Characterisation of the L-plastin Ser5 phospho antibody and expression of wild-type L-plastin and phosphorylation variants in Vero cells. (A) Schematic representation of wild-type L-plastin protein (WT) and of phosphorylation variants, and a modular arrangement of L-plastin domains. The amino-acid sequences of the wild-type phosphorylation sites (Ser5 and Ser7) and of the sv-tagged phosphorylation variants (S/A, S/E) are indicated. (B) The anti-Ser5-P antibody reacts specifically with in vitro phosphorylated recombinant L-plastin. Equal amounts of recombinant wild-type L-plastin (WT) or or L-plastin Ser5Ala (S/A) were incubated with the catalytic domain of protein kinase A for various time points as described in Materials and Methods. L-plastin was analysed by immunoblotting with anti-Ser5-P (upper panel) or anti-L-plastin antibodies (lower panel). (C) The anti-Ser5-P antibody specifically reacts with phosphorylated L-plastin in Jurkat T lymphoid cells. Jurkat cells were stimulated with 1 mM 8-Bromo-cAMP or 0.1 mM forskolin for 45 minutes. Before stimulation, cells were treated (+) or not (-) with 50 µM of H-89 for 45 minutes. Equal amounts of cell lysates were analysed by immunoblotting with anti-Ser5-P (upper panel) or anti-L-plastin antibodies (lower panel). (D) Expression and Ser5 phosphorylation of wild-type L-plastin and phosphorylation variants in Vero cells. Vero cells were transfected with cDNA constructs encoding sv-tagged wild-type (WT), Ser5Ala (S/A) or Ser5Glu (S/E) L-plastin. Untransfected cells (UT) were used as a negative control. After 48 hours, equal amounts of cell extracts were analysed by immunoblotting. Transfected proteins were detected with anti-sv-tag (upper panel) or anti-Ser5-P antibodies (lower panel).