EGF-induced αDGK activation in NIH-EGFR/ALK cells. NIH-EGFR/ALK cell line was transiently transfected with the expression vector pMT2-myc-αDGK according to the calcium-phosphate technique. Cells were serum-starved for 18 hours and then either mock-treated (-) or stimulated (+) for 10 minutes at 37°C with 100 ng/mL EGF and lysed thereafter. Lysates were immunoprecipitates with appropriate concentrations of agarose-conjugated anti-c-myc antibody. (A) Immunocomplexes were subjected to in vitro kinase assay as described in Figure 1. (B) An aliquot of each sample was analyzed by Western blot to control that equal amounts of myc-αDGK were present during in vitro kinase assay.