Effect of Unc119 G22V on activation of Lck and its downstream target ERK1/2. (A) CD4 T cells were isolated from PBMCs obtained from a healthy donor. To facilitate retroviral infection, cells were stimulated with plate-bound anti-CD3 and anti-CD28 Abs for 48 hours. Cells were then infected with bicistronic retroviruses encoding GFP alone (EV, empty virus) or GFP and wild-type Unc119 (WT) or the G22V mutant of Unc119 (G22V). GFP⁺ cells were sorted, lysed, and blotted with anti-Unc119 and anti-actin Abs. The viral Unc119 and its mutant had a FLAG tag attached to their C terminus. Therefore, corresponding bands have a higher molecular weight compared with the endogenous Unc119 (endo Unc119) band and are shifted upward. (B) Infected/sorted primary CD4 T cells were stimulated as in Figure 1 and lysed. Lysates were subjected to immunoprecipitation with an anti-FLAG Ab. Immunoprecipitates were run on polyacrylamide gels and sequentially blotted with anti-Lck and anti-FLAG Abs. IgH indicates Ig heavy chain. (C) Infected/sorted primary CD4 T cells were analyzed for Lck activity as in Figure 1. (D) Infected/sorted CD4 T cells were stimulated as in Figure 1 and lysed. Lysates were blotted with anti-phospho-ERK1/2 (T202/Y204) and anti-ERK1 Abs. Panels A through D are each representative of 3 independent experiments performed using CD4 T cells from 3 different healthy donors.