Fig. 4. ARAP3 is tyrosine phosphorylated during attachment of HEK293 cells to the ECM substrate fibronectin. (A) Diagram of ARAP3 proteins introduced into tet-inducible Flp-In^TM T-REx^TM HEK293 cell lines. Crosses designate domains with mutated `catalytic arginine' residues required for GAP activity. (B) Stable Flp-In^TM T-REx^TM HEK293 cell lines (pools of >20 isogenic clones) harbouring stably integrated cDNAs for the indicated ARAP3 proteins were treated (+) or not treated (-) with 2 µg/ml doxycycline (DOX) for 16 hours. Cell lysates (20 µg protein) were analysed by SDS-PAGE and western blotting (WB) with antibodies against ARAP3, p120^RasGAP and p85 (the PI 3-kinase regulatory subunit). (C) HEK293 Flp-In^TM cell lines treated for 16 hours with doxycycline to induce expression of the indicated ARAP3 proteins were trypsinised, washed and resuspended in serum-free medium for 1 hour. Suspension cells (0 minutes) or cells plated onto fibronectin (FN)-coated plates for the indicated times were lysed and immunoprecipitated with anti-FLAG then subjected to SDS-PAGE and western analysis with anti-phosphotyrosine (pY) then anti-FLAG antibodies (right panels). (D) The indicated HEK293 Flp-In^TM cell lines were treated as in C. Suspension cells were pre-incubated with 15 µM LY294002 (+) or DMSO (-) for 20 minutes and the cells plated onto fibronectin for 30 minutes. FLAG-ARAP3 proteins were analysed as described in C. (E) Serum-starved 293T cells expressing ARAP3 were harvested and held in suspension for 1 hour then incubated for 30 minutes with the indicated concentrations of the Src inhibitors PP2 and SU6656 and the negative control compound PP3. Cells were plated onto fibronectin-coated plastic (10 µg/ml) for 30 minutes and lysed. Samples were analysed by western blotting with phosphotyrosine and ARAP3 antibodies. (F) 293T cells were transfected with plasmids encoding ARAP3 (2 µg) and the indicated amounts of a dominant interfering Src mutant (Src KD). Cells were plated on fibronectin for 30 minutes and samples of the cell lysate analysed as described in E.