Figure 1: Effects of ITK-Y511F and ITK-BTK(SH3) mutations on ITK phosphorylation. (a) Jurkat cells that had been transfected with the indicated ITK constructs were stimulated (+) or not (−) with anti-CD3ε antibodies and then lysed. ITK in the lysates was immuno-precipitated (IP) with anti-ITK antibodies, immune complexes resolved by SDS-PAGE, proteins transferred onto PVDF membranes and immuno-blotted (IB) sequentially with antiphosphotyrosine antibodies and anti-ITK antibodies as indicated. Bands were visualized by chemiluminescence as described in Section 2. Numbers under the panels represent the fold increase in band signal intensity (phosphorylation) compared to the respective nonstimulated control calculated as described in Section 2. Results are those from one of three replicate experiments with similar results. (b) Murine thymocytes from ITK-deficient mice, nucleofected with the indicated ITK constructs, were stimulated with anti-CD3ε antibodies for 1 minute and then analyzed for ITK phosphorylation by flow cytometry using Alexa 647-tagged antibodies that react with ITK pY511, 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. (c) Average (±SEM) percentage of pY511 positive cells of three replicate experiments (including the experiment depicted in panel (b) performed as described in panel (b). The * denotes that the average of pY511 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-pY511 reactivity.