Figure 4.  WASp inducibly associates and colocalizes with PSTPIP1 and PTP-PEST. (A) Jurkat T cells were stimulated for the indicated times with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-PSTPIP1 antibody. The immune complexes were subjected to SDS-PAGE and sequentially immunoblotted with anti-PST-PEST, anti-WASp, and anti-PSTPIP1 antibodies. (B) Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-WASp antibody. Complexes were resolved by SDS-PAGE followed by sequential immunoblotting with anti–PTP-PEST and anti-WASp antibodies. (C) Lysates prepared from Jurkat T cells were incubated with GST, GST-PSTPIP1, full-length (FL), GST-PSTPIPCOIL, or GST-PSTPIPSH3 fusion proteins bound to glutathione- sepharose beads. Complexes were resolved by SDS-PAGE and immunoblotted using an anti–PTP-PEST antibody. (D) Cos-7 cells were transiently transfected with pEGFP-PSTPIP1 (a), pEGFP-PTP-PEST (b), pEGFP-WASp (c), pEGFP-PSTPIP1 and pcΔNA3-PTP-PEST (d), or pEGFP-PSTPIP1, DSRED-WASp, and pcDNA3-PTP-PEST (e). Cells were fixed, stained with rhodamine phalloidin for actin (a–c) or with anti–PTP-PEST antibody (d and e) and Cy5 anti–rabbit Ig (e), and then analyzed by confocal immunofluorescent microscopy. The images shown are representative of three independent experiments. (E) pcDNA3 constructs for expression of wild-type or catalytically inactive (C231S) PTP-PEST were cotransfected with pEGFP-WASp (WASp-GFP) into Jurkat cells. The cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies, lysed, and the lysate proteins were immunoprecipitated with anti-GFP antibodies. The complexes were subjected to SDS-PAGE and immunoblotted sequentially with anti–p-Tyr and anti-GFP antibodies.