FIG. 3.

Mitotic phosphorylation and activation of Src. (A) A fraction of the lysates used for RPTPα immunoprecipitation (Fig. 2A) was boiled in SDS sample buffer, and the samples were run on a 7.5% SDS-polyacrylamide gel. The proteins were transferred to PVDF membranes, and the membranes were probed with anti-pY416 antibody and subsequently, after stripping, with anti-npY527 and anti-Src. (B) Endogenous Src was immunoprecipitated with cross-linked antibodies to protein A beads from unsynchronized and mitotic NIH 3T3 cells. Half of the immunoprecipitate was subjected to an in vitro kinase assay, using enolase as substrate. The other half was used for immunoblotting with anti-Src antibody followed by enhanced chemiluminescence (ECL) (bottom panel). The amount of incorporated phosphate was visualized by autoradiography (top panel). The positions of enolase and Src are indicated by arrows. WCLs, whole-cell lysates; IB, immunoblot; IP, immunoprecipitation.