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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: J Immunol. 2012 Jun 25;189(3):1154–1161. doi: 10.4049/jimmunol.1200123

Figure 4.

Figure 4

Themis1 and Themis2 are tyrosine phosphorylated by src kinases following TCR or BCR engagement. A, Analysis of Themis1 and Themis2 phosphorylation in thymocytes following stimulation with anti-CD3+anti-CD4 antibodies or of Themis2 phosphorylation in B cells following stimulation with F(ab’)2 anti-IgM antibodies. B, Analysis of Themis1 and Themis2 phosphorylation by Src or ZAP70 kinases in 293T cells. 293T cells were transfected with expression plasmids encoding Flag-tagged Themis1 or Themis2 and myc-tagged tyrosine kinases [wild-type Lck, active-Lck* (Lck Y505F), Fyn, Zap70]. Themis1 and Themis2 were immunoprecipitated with anti-Flag resin and immunoblotted with anti-phosphotyrosine. Whole cell lysates (WCL) were also analyzed by immunoblotting with anti-myc antibodies. C, Jurkat T cells deficient or not for Lck (JCam1.6) or ZAP70 (P116) were stimulated with pervanadate for the indicated periods of time. Themis1 was immunoprecipitated and immunoblotted with anti-phosphotyrosine. Results shown are representative of three experiments.