Fig. 4.

FGF19-activated Src kinase mediates Y67-FXR phosphorylation. a PMH from FXR-KO were transfected with siRNA for Src, ALK, JAK2, and INSR and infected with Ad-flag-FXR, and then the cells were treated with 50 ng ml⁻¹ FGF19 for 10 min. Levels of indicated proteins were detected by IB. b PMH were treated with vehicle or FGF19 for the indicated times, and activated p-Y416-Src levels were determined by IB. c Mice were fed a ND or 0.5% CA chow for 3 h, and the interaction of FXR with Src was detected by CoIP (n = 3). d Fragments of FXR (left) and c-Src (right) that were fused to GST (top). Binding of Src or flag-FXR to GST-FXR or GST-Src fusion proteins, respectively, was detected by IB. e Flag‐FXR‐WT and siRNA for Src were expressed in hepatocytes which were treated with FGF19 for 10 min. p-Y-FXR and p-Y67-FXR levels were detected by IP/IB. Relative p-Y-FXR levels are shown on the bottom (n = 3). f C57BL/6 mice were infected with lenti-shRNA for Src or control RNA for 1 month, and mice were briefly fed a 0.5% CA chow, and then, p-Y67-FXR levels in liver extracts were detected by IB (n = 2 mice). g Flag-FXR-WT or Y67F-FXR was expressed in HepG2 cells, purified FXR proteins were incubated with ATP and recombinant Src (ab60884, Abcam) as indicated, and p-Y67-FXR levels were detected by IB. h Liver sections from C57BL/6 mice or FGF15-KO mice that had been fasted (Fs) or refed (Fd) were performed by IHC using the p-Y416-Src antibody. Scale bar, 50 µm. i–m C57BL/6 mice were infected with lentivirus expressing shRNA for Src for 2 weeks and treated for 48 h with ANIT or vehicle. i Experimental outline. j Protein levels of Src detected by IB. k Serum samples. l Liver sections stained with H&E or F4/80 antibody. Scale bar, 300 µm (H&E) and 100 µm (F4/80). m Levels of the hepatic and serum BAs and total bilirubin, AST, and ALT in serum. All values are presented as mean ± SD. Statistical significance was measured using the e one- or m two-way ANOVA with the Bonferroni post-test. *P < 0.05, **P < 0.01, and NS, statistically not significant