Figure 2: Reduced transphosphorylation of PLCγ1 by ITK-Y511F and ITK-BTK(SH3) mutants. (a) murine thymocytes from ITK-deficient mice, nucleofected with the indicated ITK constructs, were stimulated with anti-mouse-CD3ε antibodies and then analyzed for PLCγ1 phosphorylation using Alexa 647-tagged antibodies that react with PLCγ1 pY783, as described in Section 2. Results are displayed as cell number (linear scale) versus fluorescence intensity (logarithmic scale). In each panel, the open histograms represent nucleofected/nonstimulated cells, the black filled histograms nonnucleofected/stimulated cells, and the grey filled histograms nucleofected/stimulated cells. The cDNA’s used in each nucleofection is indicated to the right of each panel. The vertical line denotes the electronic gate used to calculate percent positive cells. The results are those of one representative experiment using the same cells as in Figure 1(b). (b) Average (±SEM) percentage of pY783 positive cells of three replicate experiments (including experiment in panel (a) performed as described in panel (a). The cells used in these experiments are the same as those used in Figure 1(c). The * denotes that the average of pY783 positive cells in WT-ITK nucleofected group is significantly different from the rest of the groups at (Student’s t test). The dotted line demarcates the specific anti-pY783 reactivity. (c) Average (±SEM) percentage of pY319 positive cells of four replicate experiments performed as described in panel (a), but analyzed for ZAP-70 phosphorylation using Alexa 647-tagged antibodies that react with ZAP-70 pY319.