Association of αDGK protein and activity with p60^src in NIH-EGFR/ALK and NIH-EGFR/ALK-Y979F cells. (A-B) NIH-EGFR/ALK and NIH-EGFR/ALK-Y979F cells, expressing comparable levels of receptors, were serum-starved for 18 hours and then either mock-treated or stimulated for 5 minutes at 37°C with 100 ng/mL EGF and lysed thereafter. (A) A 2-mg aliquot of total cellular proteins from each lysate was incubated with GST-p60^src/SH2 (10^-7 M) immobilized onto glutathione-agarose for 1.5 hours at 4°C. After extensive washing, proteins were electrophoresed on SDS-PAGE and immunoblotted with the anti-ALK monoclonal antibody (top panel). The same lysate (100 μg) was directly analyzed by immunoblot with the anti-ALK antibody (bottom panel). (B) Total cellular proteins (3 mg) were immunoprecipitated with an anti-p60^src-specific antibody. Two thirds of each immunoprecipitate was subjected to in vitro DGK enzymatic activity as described in Figure 1 (top panel). The remainder of each immunoprecipitate was analyzed by immunoblot with p60^src antibody (bottom panel). Comparable results were obtained in 3 different experiments. (C) NIH-EGFR/ALK and NIH-EGFR/ALK-Y979F cells were transiently transfected with the expression vector pMT2-myc-αDGK. Cell treatment and lysis were performed as described in Figure 3. Control (top panel) and p60^src immunoprecipitates (middle panel) were analyzed for myc-αDGK protein by Western blot with anti-myc antibodies. Each lysate was also analyzed for proper receptor activation by immunoprecipitation with Ab-1 antibody, which recognizes the EGFR extracellular domain and subsequent Western blot with anti-phospho-Tyr antibodies (bottom panel). WB indicates Western blot.