Figure 7. TRAF3 is a proximal signaling component of TCR and CD28.

(A) Splenic pan T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice by negative selection, and Treg cells were depleted using anti-CD25 magnetic beads. Cells were stimulated with anti-CD3, or anti-CD3+anti-CD28, followed by a crosslinking Ab as described in the Methods at 37°C for indicated time periods. Total cellular lysates were immunoblotted for phosphorylated (P-) or total ERK, LAT, PLCγ1, ZAP70, and IκBα, followed by TRAF3. (B) Splenic naive Thy1⁺CD25⁻CD44^low T cells were sorted from 2–3 month-old LMC and T-TRAF3^−/− mice using a BD FACS Aria II. Cells were stimulated with anti-CD3+anti-CD28 Abs, followed by a crosslinking Ab as described in the Methods at 37°C for indicated time periods. Total cellular lysates were immunoblotted for phosphorylated (P-) or total ERK, LAT, PLCγ1, and ZAP70, followed by TRAF3. (C) Splenic pan T cells were purified from 2–3 month old LMC mice by negative selection. Cells were stimulated with anti-CD3, anti-CD28, or anti-CD3 + anti-CD28 mAbs, followed by Dynabeads coated with a crosslinking Ab as described in the Methods at 37°C for 3 minutes. The immunoprecipitates were analyzed by immunoblotting for TRAF3, Malt1, TRAF6 and ZAP70. (D) Splenic pan T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice by negative selection. Cells were stimulated with anti-CD3 + anti-CD28 mAbs, followed by Dynabeads coated with a crosslinking Ab as described in the Methods at 37°C for indicated time periods. The immunoprecipitates and residual lysates after immunprecipitation were analyzed by immunoblotting for TRAF3 and ZAP70, followed by Actin.