Figure 2. Generation of transgenic mice expressing an allele sensitive mutant of Itk.

A) Analysis of tyrosine phosphorylation of immunoprecipitated WT Itk, Itk F⁴³⁴G or Itk F⁴³⁴G/Δ429 (Itkas) following transfection into HEK293T cells (left panels). Effect of indicated concentrations of 1-NM-PP1 on tyrosine phosphorylation of WT Itk, Itkas or related kinases Tec and Txk or in transfected cells (right panel), representative of at-least 2 independent experiments. B) Effect of increasing concentrations of 1-NM-PP1 (1 nM to 10 µM) on tyrosine phosphorylation of PLCγ1 by WT Itk or Itkas. Total cell lysates probed with phospho- and total PLCγ1. Expression of Itk was confirmed, representative of at-least 2 independent experiments. C) Quantification of western analysis of Itk expression in thymocytes from WT or Tg(CD2-hItkas)Itk^−/− mice (left panel). Quantification of Itkas mRNA in purified CD4⁺ and CD8⁺ T cells from Tg(CD2-hItkas)Itk^−/− mice, expression level in CD4⁺ T cells set as 1 (right panel), Values are means ± SEMs of n=3. D) Thymocytes were analyzed for CD4 and CD8 SP cells by FACS and E) percentages determined by gating on TCRβ⁺CD4⁺CD8⁻ (CD4SP) TCRβ⁺CD4⁻CD8⁺ (CD8SP) cells. F) Percentage of PLZF⁺ thymocytes. Values are means ± SEMs of n≥5, *p < .05 by unpaired student t test.