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. Author manuscript; available in PMC: 2010 Sep 17.
Published in final edited form as: Sci Signal. 2010 Aug 3;3(133):ra59. doi: 10.1126/scisignal.2000976

Fig. 6.

Fig. 6

Effects of knockdown of Duox1 on TCR signaling in CD4+ human T cell blasts. CD4+ human T cell blasts were transfected with NT or Duox1-specific siRNAs and cultured for 40 hours. (A) Effects of NT or different Duox1-specific siRNAs (Duox1-2 or Duox1-3) on the abundances of Duox1 and Duox2 proteins. (B) Effects of NT or Duox1-specific siRNAs on TCR-induced oxidation of DCFDA. The data represent the mean ± SEM of four separate experiments. *P < 0.05. (C and D) Western blotting analysis was performed on post-nuclear cell lysates for the presence of (C) tyrosine phosphorylation, with an antibody against phosphotyrosine (pY) residues, or (D) phosphorylation of ZAP-70 (Y319) in cells stimulated for 5 min by TCR cross-linking. (D) PLC-γ1 was immunoprecipitated from the respective cell lysates and analyzed by Western blotting with a phosphospecific (Y783) antibody. Western blots were then stripped and analyzed for the presence of ZAP-70, Lck, Grb2, and PLC-γ1 as loading and siRNA controls. (E) Effects of knockdown of Duox1 on cytokine production. Cells transfected with the siRNAs described in (A) were stimulated for 8 hours with plate-bound OKT3, and supernatants were analyzed by multiplex bead arrays for the secretion of the indicated cytokines. The data represent the mean ± SEM of duplicate wells from three separate experiments. *P < 0.05.