Fig. 5. In vitro binding of unsynchronized and mitotic PTPα to the Grb2 and Src SH2 domains. (A) Lysates from unsynchronized (U, odd lanes) or mitotic (M, even lanes) PTPα-HA overexpressor cells were affinity-precipitated by incubation with GST (lanes 3 and 4), a GST–Grb2 SH2 domain fusion protein (lanes 5 and 6) or a GST–Src SH2 domain fusion protein (lanes 7 and 8) bound to Sepharose beads. The washed beads were then analyzed by 9% SDS–PAGE and anti-PTPα immunoblotting. For comparison, lanes 1 and 2 (WCL) contained 0.033 times the amount of complete whole cell lysate. (B) PTPα-HA was immunopurified from unsynchronized or mitotic overexpressor cells and affinity-precipitated by GST or the GST–SH2 domain fusion proteins used above, analyzed by 9% SDS–PAGE and immunoblotted with anti-PTPα antibody (lanes 3–8). For comparison, lanes 1 and 2 (Total) contain 0.3 times the amount of immunopurified PTPα used in the affinity precipitations. The positions of molecular weight standards are indicated in kDa.