FIGURE 5.

Dbs increases the phosphorylation of p130^Cas and its association with Crk in a Cdc42- and Ack1-dependent manner. A, T47D cells stably expressing onco-Dbs, proto-Dbs, or cognate vector were cultured on collagen I. Lysates were prepared and separated into two aliquots. The first aliquot was subject to immunoprecipitation (IP) using an anti-p130^Cas antibody. The immunoprecipitate was then examined by Western blot (WB) for the presence of p130^Cas and Crk. The second aliquot was then examined by Western blot for levels of p130^Cas phosphorylated on Tyr-410 (p-p130^Cas), total p130^Cas, Crk phosphorylated on Tyr-221 (p-Crk), and total Crk, as indicated. B and C, the stable cell lines were cultured on collagen I and transfected with siRNAs (si) targeting Cdc42 and Ack1. Scrambled siRNAs were included as controls (si Ctrl). Lysates were collected, and one aliquot was examined by Western blot for expression of Cdc42 and Ack1 (B). C, an additional aliquot of the lysate was then examined for levels of phosphorylated (p-p130^Cas) and total p130^Cas. A third aliquot was subject to immunoprecipitation, and the immunoprecipitate was examined by Western blot with an anti-Crk antibody.