Fig. 6

Src is a tyrosine kinase of p110α. A The tyrosine kinases of p110α Y317 and Y508 were predicted by GPS 3.0 and NetPhos 3.1. B Schematic of isogenic DLD1 p110α Y317F FLAG tagged cell line. DLD1 p110α 3×FLAG tagged cell line [23] were endogenously knocked in Y317F mutation on PIK3CA locus. C p110α interacts with Src. DLD1 cells, DLD1 p110α FLAG tagged cells, or DLD1 p110α Y317F FLAG tagged cells were lysed and immunoprecipitated with anti-FLAG agarose beads. The immunocomplex were blotted with indicated antibodies. D Y317 or Y508 mutation block the interaction between p110α and Src. HEK293T cells were transfected with indicated plasmids. Cells lysates were immunoprecipitated with anti-FLAG agarose beads, followed by Western blot analysis with indicated antibodies. The FLAG-tagged p110α constructs included wild-type p110α (WT); p110α Y317F mutant protein (Y317F); p110α Y508F mutant protein (Y508F); and p110α Y317F Y508F double mutant protein (Y317F/Y508F). E In vitro tyrosine kinase assay by Phospho-tag SDS-PAGE strategy. p110α/p85α protein complex were purified with Bac-to-Bac baculovirus expression system. Src proteins were purified with bacterial protein expression system. p110α and Src proteins were incubated at 37℃ in tyrosine kinase buffer for indicated time and reactions were stopped by boiling samples. Samples were dissolved with phospho-tag SDS-PAGE to detect p110α phosphorylation, or with SDS-PAGE gel to quantify the loading amounts of proteins. F Src inhibitor suppresses phosphorylation of p110α. p110α/p85α protein complex and Src recombinant proteins were incubated with or without Src inhibitor Saracatinib. p110α phosphorylation were detected with Phospho-tag SDS-PAGE strategy. G Src inhibition suppresses phosphorylation of MLC2 and AKT. Indicated cell lines were treated with Saracatinib for 36 h and then cell lysates were blotted with indicated antibodies. H-I Src IHC staining were performed with CRC tissue microarray (375 CRC patients samples from Renji Hospital). Src staining were showed in (H) and overall survival were calculated with Kaplan-Meier survival analysis (I)