Anti rock1

Liver tissues were fixed in 10% formalin overnight at 4 °C and then embedded in paraffin followed by slicing into 5 μm thick sections. The liver slices were mounted on the glass and dried in the oven for 1 h at 60 °C. Slices were deparaffinized by washing with xylene and rehydrated through a graded concentration of alcohol. The tissue sections were washed with TBST and then blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature followed by incubation with primary antibody (anti-ROCK1; 1: 1,000; PA5-22262, Invitrogen, USA) overnight at 4°C. The next day the primary antibody was removed and PBS was used to wash the slices. Secondary antibodies were added to incubate the slices for an additional 1 h at room temperature. Slices were washed again with PBS and then a commercial kit (Abcam, USA) was used to detect the signal following the guidance of the manufacturer. A light microscope was used to take images of stained slices.

Human iPSCs were washed with cold PBS, incubated for 10 min on ice in RIPA Buffer (Sigma-Aldrich), and supernatant collected. Three replicates were used for each condition. The supernatant protein content was determined using a Pierce BCA Protein Assay kit (Thermofisher Scientific) colorimetric reaction and quantified on a SpectraMax i3 Multi-Mode Platform (Molecular Devices). Subsequently, 20 μg of protein from each sample was resolved by SDS-PAGE, and transferred to a nitrocellulose membrane (Invitrogen). The membranes were incubated overnight at 4°C with primary antibodies: anti-ROCK1 (AbCAM 1:200), anti-CDH1 (AbCAM 1:200), anti-GAPDH, (Invitrogen 1:10,000), followed by incubation (30 min at room temperature) with infrared secondary antibodies: IRDye 800CW and IRDye 680CW (LI-COR 1:13,000), and imaged on the Odyssey Fc Imaging System (LI-COR Biosciences). Protein levels were quantified using Image Studio Lite (LI-COR Biosciences).