Human β actin

To indirectly assess the activation/inhibition of RhoA/ROCK signaling, the protein expression of p-MBS and t-MBS was detected by western blotting. The protein was extracted from the peripheral blood samples, and the protein concentration was determined using a bicinchoninic acid (BCA) protein detection kit (Beyotime, Shanghai, China). The proteins were then separated by electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skimmed milk, and incubated overnight at 4°C with antibody against human MBS, human p-MBS, or human β-actin (Santa Cruz, USA). The membranes were then incubated with secondary antibody, and the proteins were visualized using enhanced chemiluminescence reagent (Beyotime). Finally, a ChemiDoc™ XRS + Imaging System (Bio-Rad, USA) was used to take photos of the membranes, and the results were quantified to calculate the p-MBS/t-MBS ratio in each sample.

Whole-cell extracts (see
Cell culture), scraped out and extracted using RIPA Lysis and Extraction Buffer, were run on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus following the standard protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with rabbit antibodies against human ABCB11 (Affinity, Catalog #DF 9278) 1: 2000 dilution; human β-actin (Santa Cruz Cat.# SC4778), dilution 1:1000; 4°C overnight. The membrane was washed with TBST buffer and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit (Santa Cruz Cat# SC-2004)/anti-mouse antibodies (Cat.#SC-2005) for 2 h at room temperature. Blots were washed with TBST four times and developed with the ECL system (Bio-Rad, US Cat.#170-5060) according to the manufacturer’s protocols. Raw, uncropped images from western blotting are available as Underlying data
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