Figure 7.

Association followed by dissociation of ITK to TSAD PRR is required for maximal phosphorylation of ITK in the presence of LCK. A, immunoprecipitation experiment showing phosphorylation of ITK in the absence or presence of intact or mutated TSAD molecules. 293T cells were transfected with the indicated cDNA plasmids. Myc-tagged ITK proteins were immunoprecipitated from the cell lysates, and the level of phosphorylation was assessed by immunoblotting. The result is one representative of two experiments. B, the graph shows quantitation of signal densities using ImageJ of the experiment shown in A (n = 4, mean ± S.D. (error bars)). C and D, immunoprecipitation experiments as in A, including also a Myc-ITK-Y180F mutant (n = 3, mean ± S.D.). E–G, immunoprecipitation experiments in 293T cells expressing the indicated plasmids immunoblotted with the indicated antibodies. F, graph shows quantitation of signal densities using ImageJ of the experiment shown in E and shows the relative amount of pTyr⁵¹¹ signal where the pTyr⁵¹¹ signal in ITK is set to 1 (n = 5, mean ± S.D.). H, the graph shows quantitation of signal densities using ImageJ of the experiment shown in G and shows the relative amount of ITK co-immunoprecipitated with TSAD (n = 5, mean ± S.D.).