Fig. 6

L-plastin Ser5 phosphorylation is not required for L-plastin/cortactin interaction. a L-plastinEF-ABD1 is phosphorylated upon PMA stimulation. HEK 293T cells were transfected with L-plastinWT-GFP or L-plastinEF-ABD1-GFP. Cells were treated with or without 0.1 μM PMA for 1 h and whole-cell lysates were submitted to L-plastin (red) and Ser5-P-L-plastin (green) immunoblotting. b Co-immunoprecipitation of cortactin with L-plastinWT in HEK 293T cells. Cells were transfected with GFP, L-plastinWT-GFP or L-plastinEF-ABD1-GFP and treated with 0.1 μM PMA for 1 h. Following cell lysis, protein extracts were subjected to immunoprecipitation with GFP-nanotrap. Aliquots of input [In], non-bound [NB], and bound [B] fractions were separated by SDS-PAGE and proteins were visualized by immunoblotting using an anti-cortactin antibody (#05-180). In the bound fraction, an intense unspecific signal is visible that corresponds to the important amount of precipitated L-plastinWT-GFP or L-plastinEF-ABD1-GFP. C) Co-immunoprecipitation of cortactin with L-plastinWT and L-plastin phosphovariants. Cells were transfected with GFP, L-plastinWT-GFP, L-plastinS5E-GFP and L-plastinS5A-GFP and treated with or without 0.1 μM PMA for 1 h. Protein extracts were subjected to immunoprecipitation with GFP-nanotrap. Aliquots of input [In], non-bound [NB], and bound [B] fractions were separated by SDS-PAGE and proteins were visualized by immunoblotting using an anti-cortactin antibody (#sc-11408). In the bound fraction, an intense unspecific signal is visible that corresponds to the important amount of precipitated L-plastin-GFP variants