Bicinchoninic acid protein quantitative kit

The cultured cells were harvested by trypsin digestion and lysed with enhanced Radio-Immunoprecipitation assay cell lysis buffer encompassing protease inhibitor (BOSTER, Wuhan, Hubei, China). The protein concentration was estimated by a bicinchoninic acid protein quantitative Kit (BOSTER). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted to a polyvinylidene fluoride membrane that was sealed at room temperature for 2 h with 5% bovine serum albumin to block nonspecific binding. Overnight probing was implemented with primary antibodies (1:1000, Cell Signaling Technology, Beverly, MA, USA) to SMURF1 (#2714), GAPDH (#5174), UVRAG (#13115), ATG5 (#12994), Wnt5a (#2392), and Ub (#5174) at 4 °C, followed by 1 h reprobing with horseradish peroxidase-tagged goat anti-rabbit IgG (ab205719, 1:2000, Abcam) at room temperature. Then the membrane was incubated for 1 min with electrogenerated chemiluminescence (ECL) working solution (EMD Millipore Corp., Billerica, MA, USA). Subsequent to discarding of excess ECL reagent, the membrane was sealed with plastic wrap, and exposed with X-ray film in the dark box for 5–10 min before development and fixation. The image J software was adopted for gray value quantitative analysis of protein bands with GAPDH as a normalizer.

The cultured cells were collected and lysed with enhanced Radio‐Immunoprecipitation assay cell lysis buffer containing protease inhibitor (Boster Biological Technology Co., Ltd.). Then, the bicinchoninic acid protein quantitative kit (Boster Biological Technology Co., Ltd.) was adopted to estimate the protein concentration. The protein was transferred to a polyvinylidene fluoride membrane after 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. After 2‐h sealing with 5% bovine serum albumin at room temperature, the membrane was probed with primary rabbit anti‐human antibodies (Abcam) to HDAC2 (ab32117, 1:2000), HNF4A (ab92378, 1:1000), ARID1A (ab182560, 1:1000), and GAPDH (ab8245, 1:1000) overnight at 4℃. Horseradish peroxidase‐labelled goat anti‐rabbit immunoglobulin G (IgG) (ab6721, 1:5000, Abcam) was added into the membrane. After 1‐h incubation at room temperature, the membrane was developed with electrogenerated chemiluminescence working solution (EMD Millipore) and exposed using a Bio‐Rad automatic imager. ImageJ analysis software was adopted to quantify the grey value of each band in the Western blot image with GAPDH as a normalizer.