Figure 7: Effects of downregulation of RanBP2 in BA regulation in mice.

(a–d) Experiments without biliary insults. (a) Experimental Outline. (b) In vivo SUMO assay: SUMO-SHP levels in RanBP2-downregulated mice were detected by IP/IB. Endogenous SHP in liver extracts was immunoprecipitated with SHP antibody and SUMOylated SHP in the immunoprecipitates was detected by IB using SUMO2 antibody. Levels of RanBP2 or SHP in liver extracts after expression of control or RanBP2 shRNA were detected by IB. (c) Effects of downregulation of RanBP2 on subcellular localization of SHP in the mouse liver. SHP was detected by IHC. Scale bar, 50 μm. Effects on expression of the indicated genes (d) or the levels of BA, TG, and cholesterol (e) in the liver. In e, values for the control shRNA groups are set to 1. (f,g) Experiments with a biliary insult by BA overload. (f) Experimental outline and protein levels of RanBP2 detected by IB (top). The volume of the gall bladder, serum BA levels, liver BA levels, and serum ALT and AST levels were determined. BA levels are expressed per g of liver wet weight. Values represent mean±s.e.m. Statistical significance was determined by the Student’s t-test, (n=5 mice, *P<0.05, **P<0.005, and NS, statistically not significant) (d–f). (g) Liver sections were stained with haematoxylin and eosin (H&E) to detect necrosis and macrophages were detected (brown) with F4/80 antibody. Scale bar, 100 μm.