Figure 5. SKAP55 expression in ADAP^-/- T cells fails to rescue T:APC conjugate formation.

(A) Jurkat T cells were transduced with control adenovirus (Ctrl) or with the indicated HA-tagged ADAP constructs. After 2d, 10⁶ cells were left unstimulated or stimulated for 5 min with anti-TCR mAb OKT3, lysed, and immunoprecipitated with agarose-conjugated anti-HA and western blots for ADAP and SKAP55 performed. The relative molecular mass of size standards is shown on the right. Similar results were observed in four independent experiments. (B) Naïve hCAR/ADAP^+/+ lymphocytes expressing endogenous levels of SKAP55 were transduced with either full-length murine ADAP (wtADAP) or ADAP lacking the restricted proline rich domain (Δ338-358). Cells were lysed as described in Materials and Methods, immunprecipitated with agarose-conjugated anti-HA antibodies, and Western blots were performed with anti-HA or anti-SKAP55 antibodies. (C) Naïve DO11.10/hCAR/ADAP^-/- (KO) or ADAP^+/+ (WT) lymphocytes expressing the indicated constructs were analyzed for T:APC conjugate formation between KJ-126+/Thy1.1+ T cells and Balb/c splenocytes pre-pulsed with the indicated concentrations of OVAp. Results are shown for a single representative experiment and are representative of at least four independent experiments performed for each construct.