Figure 3: Deficient Th2 cytokine production by ITK-Y511F and ITK-BTK(SH3) mutants. Murine thymocytes were nucleofected with the indicated ITK constructs and then cultured under Th2 skewing conditions as described in Section 2. Culture supernatants were assayed in duplicate for IL-4 (a) and IL-13 (b) using commercial ELISA assay kits and following manufacturer’s recommendations. Results are displayed as the averages (±SEM) of three replicate experiments for IL-4 and two replicate experiments for IL-13. Negative controls (nonstimulated cell-culture supernatants) contained 69 pg/mL IL-4 and undetectable (<4 pg/mL) IL-13.