Effects of Itk siRNA on the phosphorylation of various signaling molecules in Jurkat cells. Jurkat cells untransfected or transfected with Itk-specific siRNA were treated with or without anti-CD3 for various times and the cellular proteins were separated by PAGE. A, The tyrosine phosphorylation of specific sites on LAT and PLC-γ1, along with the expression level of GAPDH, was determined by immunoblotting. B, The percentage of maximum phosphorylation for LAT tyrosine 132 in Jurkat cells was determined. The mean ± SEM of three separate experiments for each time point was plotted and fit using Origin. C, The percentage of maximum phosphorylation for PLC-γ1 tyrosine 783 in Jurkat cells was determined. The mean ± SEM of three separate experiments for each time point was plotted and fit using Origin. D, The expression levels of Itk and GAPDH were determined by immunoblotting in Jurkat cells untreated or treated with Itk-specific siRNA. Duplicate samples for both treatments are shown.