SHP1 Antibody - #DF6414

Fig. 2. CP-25 inhibited Th17 cell differentiation through recovering SHP1 and STAT3 interaction. (A) Representative pSTAT3Tyr705 and STAT3 immunoblotting of splenic lymphocytes from control, Veh, CP-25 or PAR treated CIA mice. (B) Statistic analysis of the ration of pSTAT3Tyr705 against STAT3 in splenic lymphocytes from 4 groups. (C) CD4 was labeled with Alexa Fluor 647 and pSTAT3Tyr705 was labeled with Alexa Fluor 488 in splenic T cells isolated from individual groups of mice. (D) The mean fluorescence intensity (MFI) of pSTAT3Tyr705 was used to indicate the relative expression level of pSTAT3Tyr705 in CD4 positive T cells. (E) SHP1 was labeled with Alexa Fluor 647 and STAT3 was labeled with Alexa Fluor 488 in splenic T cells isolated from treated CIA mouse. (F) The colocalization rate between SHP1 and STAT3 was analyzed. (G) Splenic naïve (CD4+CD62L+) T cells were stimulated with IL-6, IL-21, IL-23, TGF-β for 72 h with or without NSC87877 pretreatment, followed by a 4-hour PMA, BFA and ionomycin induction, and then IL-17A+ positive Th17 cells were sorted. The cells were gated on FSC-SSC dot plot, and then the lymphocytes were gated for analyzing the percentage of Th 17 (CD4+IL-17A+) cells. (H) The percentage of Th17 cells induced by indicated conditions was compared. Data were presented as mean ± SD from 5 mice per group. ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by post hoc Tukey’s test.

Fig. 3. SHP1 inhibited STAT3 activation in CD4+ T cells in an arrb2 dependent manner. (A) Arrb2 was stained in green and SHP1 was in red in splenic T cells from treated CIA mouse. (B) The MFI of arrb2 in CD4 positive T cells was analyzed. (C) The colocalization rate of SHP1 and arrb2 was calculated. n = 5. (D) Arrb2 in lysate of splenic T cells from treated mice was pulled down and both SHP1 and arrb2 were blotted. (E) The relative intensity of arrb2 bound to SHP1 was compared between individual groups of mice. n = 3. (F) CD4+ T cells isolated from WT or arrb2 null mice were treated with or without IL-6, IL-21, IL-23 and TGF-β, and then SHP1 and STAT3 were labeled. (G) The correlation between SHP1 and STAT3 was determined. n = 6–7. (G) Total and phosphorylated STAT3 at Tyr705 site was detected by flow cytometry in T cells from WT or arrb2-/- mice followed by indicated treatment. Cells only incubated with secondary antibody were set as isotype control to exclude non-specific staining. (H-J) The MFI of pSTAT3, total STAT3 and the ratio of pSTAT3 to total STAT3 was analyzed. n = 5. Data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by post hoc Tukey’s test.