Figure 1.

Cell-line and assay development. (a) ZAP70 KI and ZAP70 SH2 (N+C) were subcloned in front of the internal ribosome entry site (IRES), followed by GFP, in the Tet-regulated retroviral vector (pTRA-IRES-GFP). (b) After infecting tTA-expressing Jurkat (4D9#32) cells with retroviral constructs containing IRES-GFP, ZAP70 KI-IRES-GFP, or ZAP70 SH2 (N+C)-IRES-GFP, cells were left unstimulated or stimulated with anti-TCR antibody for 24 h. CD69 expression was analyzed after gating on the GFP-positive population (infected population, boxed in R1). The dashed line and the thin line on the graphs indicate cells infected with IRES-GFP (vector) before and after TCR stimulation, respectively, and the thick line indicates cells infected with ZAP70 KI-IRES-GFP (top panel) or ZAP70 SH2 (N+C)-IRES-GFP (bottom panel), both after TCR stimulation. (c) After infecting Jurkat-tTA (4D9#32) cells with retroviral vector alone or vector containing ZAP70 SH2 (N+C)-IRES-GFP, cells were cultured without (top panels) or with (bottom panels) Dox for 6 days, and then left unstimulated or stimulated with anti-TCR antibody for 24 h. The box R1 indicates GFP-positive cells. CD69 expression was analyzed for the entire cell population. The dashed line and the thin line indicate cells infected with vector before and after TCR stimulation, respectively, and the thick line indicates cells infected with vector containing ZAP70 SH2 (N+C)-IRES-GFP after TCR stimulation. (d) The Jurkat-tTA (4D9#32) cells containing different retroviral constructs (shown above the lanes) were cultured in the absence (-) or presence (+) of Dox, and whole-cell lysates were prepared. Lysates were loaded (100 μg per lane) and analyzed by western blotting using anti-ZAP70 antibody (Upstate Biotechnology, Waltham, USA). The top ZAP70 band included endogenous (- and + Dox) as well as retrovirally expressed ZAP70 (-Dox only), whereas the bottom ZAP70 band contained only retrovirally expressed truncated ZAP70 SH2 (N+C).