Figure 7. Grail negatively regulates Stat6 expression and activity.

(a) Naïve CD4⁺ T cells from WT and Grail KO mice were activated with anti-CD3/anti-CD28 in the presence or absence of IL-4 for 24 hours, and Stat6 protein expression were detected by western blot analysis. (b) Naive CD4⁺ T cells from WT and Grail KO mice were cultured for 4 days under Th2 differentiation conditions and analyzed for Stat6 and Gata3 expression by western blot analysis. The numbers in a and b depict relative immune blotting signals compared to WT after normalizing with Beta-actin levels. (c) Naive CD4⁺ T cells from WT and Grail KO mice were isolated and serum starved for 12 hours followed by activation with anti-CD3/ant-CD28 in the presence or absence of IL-4 for the indicated time points and pospho-Stat6 and total Stat6 levels were determined by western blot analysis. Beta-actin levels were determined in the same samples as loading control. The numbers depict relative immune blotting signals compared to WT anti-CD3/CD28 only treated samples after normalizing with Beta-actin levels. (d)HEK293T cells were transfected with vectors encoding Stat6, HA-Ub and either Grail or a Grail mutant. The lysates were subjected to immunoprecipitation (IP) using anti-Stat6 antibody. The blot was probed with anti-HA-HRP and re-probed for Stat6 expression. (e) CD4⁺ T cells from WT and Grail KO mice were activated with anti-CD3 and IL-4 along with MG132 (proteasome inhibitor) for 6 hrs. The cell lysates were then immunoprecipitated using anti-Stat6 antibody and the blot was probed with anti-ubiquitin or anti-Stat6 antibodies. Relative western blotting signals were calculated as a ratio of ubiquitinated and non-ubiquitinated Stat6. (f) HEK293T cells were either left untransfected or transfected with vectors encoding Stat6, Grail-GFP or both. The lysates were subjected to IP using anti-Stat6 antibody. The blot was probed with anti-GFP antibody (upper panel) and reprobed with anti-Stat6 antibody (middle panel) as a control. The WCL was immunoblotted with anti-GFP antibody (lower panel) for checking antibody efficacy. The results shown in the figures are representative blots of at least three independent experiments. P values: *<0.05, Student’s t-test was performed to detect between-group differences..