Fig. 5

K63-linked ubiquitination of BSEP is essential for CHMP5-mediated apical targeting of BSEP. a BSEP was abundantly modified with K63-linked ubiquitin (Ub) chains. Hep G2 cells were co-transfected with p3XFLAG-BSEP and HA-Ub mutants or the control plasmid pcDNA3.1 ( +) for 24 h. Proteins with HA-Ub modification were isolated by anti-HA antibodies, and then detected ubiquitinated FLAG-BSEP (Ub-FLAG-BSEP) and HA-Ub linked proteins via anti-FLAG and anti-HA antibodies, respectively. The bar graph (n = 3, mean ± SD) represents the Ub-FLAG-BSEP signals normalized to HA-Ub. Two-tailed unpaired Student’s t-test was used (**P ≤ 0.01). b K63-linked ubiquitination of BSEP is important to the plasma-membrane targeting of BSEP. Hep G2 cells co-expressing FLAG-BSEP, mCherry-CHMP5 and HA-Ub-K63 or -K63R were fractionated at the indicated times. The plasma-membrane (PM) and organelle-membrane (OM) protein fractions were immunoblotted by anti-FLAG, anti-CHMP5, and anti-Na⁺/K⁺-ATPase antibodies. Na⁺/K⁺-ATPase was a PM fractionation control. Relative FLAG-BSEP signals normalized to the PM control were shown. c Immunohistochemical staining shows the strong canalicular FALG-BSEP in Ub-K63 co-expression in vivo. FLAG-BSEP and HA-Ub-K63 or -K63R were expressed in FVB/NJ mouse livers via hydrodynamic injection. The FLAG-BSEP was visualized by anti-FLAG antibodies and NovaRed; cell nuclei were stained by hematoxylin. The bar graph (n = 1, mean ± SD) illustrates the canalicular FLAG-BSEP in Ub-K63 or -K63R expressing mouse livers. Four images per mice and one mouse per group were analyzed for the canalicular targeting index. Two-tailed unpaired Student’s t-test was used (*P ≤ 0.05). The P-value is 0.0286. d Representative immunofluorescence images demonstrate Ub-K63 is essential for CHMP5-regulated membrane trafficking of BSEP in vitro. Hep G2 cells with indicated siRNA pre-treatment were co-expressed FLAG-BSEP and HA-Ub-K63 or -K63R for temperature shift assay followed by immunostainings of FLAG-BSEP (green). Cell nuclei were stained via DAPI. The arrowhead indicates the FLAG-BSEP at the plasma membrane