p140Cap tyrosine phosphorylation on EPLYA and EGLYA sequences regulate Csk binding.

<p><b>A.</b> cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPL<b>Y/F</b>A, EGLY<b>/F</b>A) were used to transfect HEK-293 cells. Cells were treated with 100 micromolar pervanadate solution as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054931#pone-0054931-g002" target="_blank">Figure 2B</a> and extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies PY99, p140Cap, and Csk. The results are representative of six independent experiments. <b>B.</b> HEK-293 cells transfected as in A for 48 hours, were starved overnight and treated for 0, 5, 15 minutes with 20%FBS. Cell extracts were immunoprecipitated with a specific antibody to p140Cap. Immunocomplexes were analysed by western blotting using monoclonal antibodies specific for phosphotyrosines, p140Cap, Csk and Vinculin.