Figure 3.

STAT3 phosphorylated by IRAK1 regulates microRNA expression. (A) SIGIRR interacts specifically with IRAK1 and MYD88, but not TLRs in HIECs at baseline. HIEC lysates were immunoprecipitated by SIGIRR antibody, then immunoprecipitated complexes were immunoblotted with the indicated antibodies. (B) HIECs were transfected with pcDNA3–empty vector (EV), pcDNA3–SIGIRR (SIGIRR), and pcDNA3–SIGIRR plus pcDNA3-SIGIRR p168X mutant, immunoprecipitation (IP) was performed using SIGIRR antibody and analyzed by Western blot with the indicated antibodies. (C and D) HIECs were treated with the IRAK1/4 inhibitor at the escalated concentrations for 24 hours, then were lysed for Western blot (WB) and real-time PCR analysis of microRNA expression. (E) ChIP quantitative PCR values of STAT3 recruitment on microRNA promoters in HIECs treated with IRAK1/4 inhibitor for 16 hours. (F–H) SIGIRR protein models for study. (F) The far-left structure shows the full mature SIGIRR protein structure (amino acids 1–410, Q6IA17; UniProt) within a lipid membrane (cyan), with labeled domains and portions of the cell. The next model shows the full protein without the membrane and labeling in red amino acid P115 and Y168. The next 2 models show SIGIRR p.P115R and p.Y168X. (G) Molecular surface plot of the intracellular Toll-interleukin receptor domain (gray) for the full SIGIRR or SIGIRR p.Y168X interacting with MYD88 (magenta). (H) Predicted effect of the SIGIRR p.P115R mutant on the first α helix of the intracellular TIR domain. The predicted change of protein structure in SIGIRR p.P115R mutant is marked by red boxes. ∗P < .05. ACTB, actin beta; C , control.