Figure 5.

Renin Phosphorylates STAT4 to Induce IL-17 Expression

(A) Western blot analysis of HOKs transfected with empty or renin plasmids, using antibodies as indicated.

(B) Western blot analysis of STAT4 and phospho-STAT4 in HOKs transfected with empty or renin plasmids.

(C) Co-IP and Western blot analyses of cell lysates from HOKs transfected with the indicated plasmids.

(D) HOKs were transfected with empty or renin plasmids. Cytoplasmic (C) and nuclear (N) fractions were extracted and analyzed by Western blotting using indicated antibodies. Lamin C is selected to be a nuclear marker, and GAPDH serves as a cytoplasmic marker.

(E) Y693F mutation eliminated DNA binding of STAT4 in a pull-down assay. Biotin-labeled SBE probes were added into the transfected HOKs lysates to bind proteins and then pulled down by streptavidin beads. Retrieved proteins were detected by Western blot. Unlabeled SBE probe is chosen for a negative control.

(F–I) The endogenous STAT4 in HOKs was deleted by CRISPR/Cas9 system, and different plasmids were transfected into the STAT4-knockout cell line. Western blot was carried out to determine the p-STAT4 and STAT4 levels in transfected HOKs (F). Chromatin immunoprecipitation (ChIP) was performed using anti-STAT4 antibody and showed that Y693F mutation impeded STAT4 binding to the promoter region of IL-17 gene (G). Real-time PCR (H) or ELISA (I) detection of IL-17 in HOKs transfected with renin plasmids in the presence or absence of TKI.

∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus corresponding control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. renin group, n = 3. Ctrl, control; WT, wild type; Y693F, Tyr693 mutant; SBE, STAT4 binding element; TKI, tyrosine kinase inhibitor. Data were shown as means ± SD. Two-tailed Student's t test and one-way analysis of variance were used.