Fig. 8. Immunoblotting of Src and active Src in A6(1) cells with and without olomoucine. (A) Multiple scratch wounds were made in confluent cultures of A6(1) cells. Scratch-wounded cultures were incubated in the absence or presence of 15 µM olomoucine and cell lysates were prepared. Next, 50 µg whole cell lysate (WCL) was loaded into each lane of a 12% SDS-polyacrylamide gel and immunoblotted with antibodies against Src or active Src (pY416). (B) The results were quantified by densitometry and averaged (n=5). Olomoucine treatment increased the level of Src(pY416) more than 2.5 times (P=0.04). Error bars=s.e.m. (C) A6(1) cells were transiently transfected with the dominant negative construct EGFP-Cdk5T33. (Left) Whole cell lysate (50 µg) was immunoblotted with antibodies against Src or active Src (pY416). Quantification by densitometry indicated that the level of Src(pY416) was elevated 1.8±0.3 times (n=3) in transiently transfected cells. (Right) 50 µg whole cell extract was immunoblotted with anti-Cdk5 antibody to demonstrate expression of EGFP-Cdk5T33 (single arrow, Cdk5; double arrow, EGFP-Cdk5). (D) A(6)1 cells were incubated in the absence or presence of olomoucine. Cell extracts from subconfluent cultures were immunoprecipitated with anti-Cdk5 antibody and the precipitated proteins were immunoblotted for Src and active Src(pY416) (left) or Cdk5 (right). Both Src and active Src(pY416) were detected in Cdk5 immunoprecipitates (arrow). An unidentified, more rapidly migrating band was occasionally detected by this antibody but does not correspond to Src(pY416) according to manufacturer's technical data sheet. Immunoblotting with Cdk5 antibody confirmed that immunoprecipitation was effective.