Figure 6.

The roles of phosphatases PTPN2 and PTPN22 in restraining GITR signaling.A, splenic T cells isolated from littermate control (LMC) or T-Traf3^−/− mice were pretreated with DMSO (upper-left), inhibitors (5 μM) of PTPN2 (SF1670, upper-right), PTPN22 (LTV-1, bottom-left) or ERK (U0126) (bottom-right) before being stimulated via GITR for the indicated times. Whole cell lysates were subjected to SDS-PAGE and Western blot analysis. Representative blots of four similar experiments are shown. B–D, quantification of four independent experiments including representative blots shown in A. Curves represent mean ± SEM of relative band intensities, where statistical significance was determined by two-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; n.s indicates no significant difference. E, subclones of HuT28.11 or Traf3^−/− HuT28.11 T cells expressing hCD40-GITR (FLAG-tagged) were stimulated via anti-hCD40 Ab-conjugated protein G beads (see Experimental procedures) for times indicated and the hCD40-GITR signaling complex was immunoprecipitated. IPs (left) were subjected to SDS-PAGE and Western blotting for TRAF3, PTPN2 and PTPN22. Blots of whole cell lysates are shown on the right. Ctrl = IP with protein G beads conjugated with isotype control mAb. Positions of TRAF3 and PTPN2 bands are indicated by arrows. ∗ indicates the heavy chain of the IP Ab remaining in the IP sample. Blots in E are representative of ≥ 3 similar experiments. Quantification of band intensity is summarized in Fig. S4.