Seeblue rainbow marker

Cell lysates were lysed by RIPA buffer (Sigma-Aldrich, USA) with Complete Protease Inhibitor Cocktail (Roche, USA). Cell lysates were transferred to 1.5 mL tube and kept at −20 °C before use. SDS-PAGE was conducted to separate the cellular proteins. And all the cellular proteins within this study were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the information of the Pre-stained SeeBlue rainbow marker (Invitrogen, USA) loaded in parallel. The membranes were probed with the following antibodies: HMGB1 (Abcam, MA, USA), RAGE (Abcam, MA, USA), NF-κB (Abcam, MA, USA) and β-actin (Sigma, USA). The blots were detected on Kodak film developer (Fujifilm, Japan). The gradation analysis of the films was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Cell lysates were lysed by RIPA buffer (Sigma-Aldrich, U.S.A.) with Complete Protease Inhibitor Cocktail (Roche, U.S.A.). Cell lysates were transferred to 1.5 ml tube and kept at −20°C before use. SDS/PAGE was conducted to separate the cellular proteins, and all the cellular proteins within the study were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the information of the Pre-stained SeeBlue rainbow marker (Invitrogen, U.S.A.) loaded in parallel. The membranes were probed with the following antibodies: c-Ski, CD31, Vimentin, Snail, Slug, and Twist (Abcam, MA, U.S.A.), and GAPDH (Sigma, U.S.A.). The blots were detected on Kodak film developer (Fujifilm, Japan).

Cell lysates were lysed by RIPA buffer (Sigma-Aldrich, USA) with Complete Protease Inhibitor Cocktail (Roche, USA). Cell lysates were transferred to 1.5 mL tube and kept at −20°C before use. SDS-PAGE was conducted to separate the cellular proteins. And all the cellular proteins within this study were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the information of the Pre-stained SeeBlue rainbow marker (Invitrogen, USA) loaded in parallel. The membranes were probed with the following antibodies: β-catenin (Cat# E247, Abcam, USA), Collagen I (ab34710, Abcam), α-SMA (ab5694, Abcam), Histone H3 (ab1791, Abcam), β-actin (Cat# ACTN05 (C4), Abcam) and GAPDH (Cat# 6C5, Abcam). The blots were detected on Kodak film developer (Fujifilm, Japan).

Mouse liver tissues or cultured cells were lysed using RIPA buffer (Sigma-Aldrich, USA), complemented with Complete Protease Inhibitor Cocktail (Roche, USA). The lysates were transferred to 1.5 ml tube and kept at −20°C before use. SDS-PAGE was conducted to separate the cellular proteins. And all the cellular proteins within this study were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the information of the prestained SeeBlue rainbow marker (Invitrogen, USA) loaded in parallel. The membranes were probed with the following antibodies: ab10296 (for TRPV1), ab5694 (for α-SMA), ab34710 (for COL1A1), and ab8226 (for β-actin). Following incubation with primary antibodies, blots were washed for four times in TBS/Tween-20 before the 1 h incubation in goat anti-rabbit horseradish peroxidase conjugate antibody at 1 : 10000 dilution in TBS/Tween-20 containing 5% skim milk. After washing extensively in TBS/Tween-20, the blots were then processed with distilled water for detection of antigen using the enhanced chemiluminescence system. The blots were visualized with the ECL-chemiluminescent kit (ECL-plus, Thermo Scientific) and detected on a Fujifilm developer (Fujifilm, Japan).