Skip to main content
. Author manuscript; available in PMC: 2017 Oct 15.
Published in final edited form as: J Biol Chem. 2002 Mar 28;277(24):21922–21929. doi: 10.1074/jbc.M201394200

Fig. 7. Effects of Ser-to-Ala mutation and PP2A treatment on in vitro binding of PTPα from unsynchronized and mitotic cells to the Src and Grb2 SH2 domains.

Fig. 7

A, lysates (containing 400 µg of total cell protein) from unsynchronized (U) or mitotic (M) WT PTPα-HA (lanes 3–8) or PTPα(S180A/S204A)-HA (lanes 11–16) overexpresser cells were affinity-precipitated by incubation with GST (lanes 3, 4, 11, and 12), a GST-Grb2 SH2 domain fusion protein (lanes 5, 6, 13, and 14), or a GST-Src SH2 domain fusion protein (lanes 7, 8, 15, and 16) bound to Sepharose beads. The washed beads were then analyzed by 9% SDS-PAGE and anti-PTPα immunoblotting. For comparison, lanes 1, 2, 9, and 10 contained 0.025 times the amount of complete whole cell lysate (WCL) used in the affinity precipitations. B, PTPα-HA was immunopurified from unsynchronized or mitotic overexpresser cell lysates (containing 1 mg of total cell protein), incubated with (+; lanes 7, 8, 11, and 12) or without (−; lanes 5, 6, 9, and 10) the serine/threonine phosphatase PP2A, and then affinity-precipitated by the GST-SH2 domain fusion proteins used for A. For comparison, lanes 1–4 (Total) contained 0.3 times the amount of immunopurified PTPα used in the affinity precipitations; these were also incubated with (lanes 3 and 4) or without (lanes 1 and 2) PP2A. The positions of molecular mass standards are indicated in kilodaltons.