FIGURE 4.

The ADAP ΔCAR and ADAP ΔTAK mutants are each unable to restore CD3/CD28-mediated IkBα phosphorylation and degradation and p65 nuclear translocation. Control hCAR⁺ T cells (Ctrl) and hCAR⁺ ADAP^−/− T cells were transduced with adenovirus encoding Thy1.1 alone (Thy) or wild-type ADAP (WT), the ADAP ΔCAR mutant, or the ADAP ΔTAK mutant prior to CD3/CD28 stimulation for 15 or 45 min. A, lysates were immunoblotted with antibodies specific for IkBα, phospho-IκB (p-IκB), phospho-Erk (p-ERK), and Erk. B, nuclear and cytoplasmic extracts were analyzed by immunoblotting with antibodies specific for p65 and the nuclear marker lamin A/C. Cytoplasmic extracts were also probed with an anti-ADAP antibody to verify expression of ADAP.