Fig. 6

Src is over-activated in DLD1 colorectal cancer cells even though the co-expressed Csk is active. a Comparison of the specific enzymatic activity of Src immunoprecipitated from DLD1 cell lysate with that of recombinant unphosphorylated Src. Src was immunoprecipitated from DLD1 cells and its specific enzymatic activity was monitored using Src optimal peptide as the substrate. Recombinant unphosphorylated Src was used as the control. b The mRNA levels of Csk and Chk determined by qPCR. c Demonstration of phosphorylation of recombinant kinase-dead Src by Csk isolated from DLD1 cells. Csk was immunoprecipitated from the lysate of DLD1 cells and its activity was monitored using Src (K295M) as the substrate. The immunoprecipitated recombinant Csk was used as the control. The presence of Csk in the immunoprecipitates was monitored by Western blotting (upper panel). Src (K295M) (0.25 μM) was incubated with the immunoprecipitatees in the presence of [γ-³²P] ATP (250 μM) and assay buffer. At the designated time points, aliquots of the reaction mixture were taken out and subsequently analysed by SDS-PAGE followed by autoradiography. The reaction mixtures containing Src (K295M) only were the negative controls. Autoradiogram images were analyzed using ImageJ to obtain the relative density value of each band