Anti mouse igg

Hepatic proteins were extracted and quantified by BCA assay kit (Boster, Wuhan, China). The experiment was constructed as previously published method [13 ]. Briefly, equal amounts of proteins were electrophoresed andelectrotransfered. Antibodies specific for ALDH1B1 (LSBio, Seattle, USA), ALDH2 (Abcam, UK), ALDH7A1 (Proteintech, Chicago, USA) and GAPDH (CST, USA) were used. Anti-mouse IgG and anti-rabbit IgG (Yeasen, Shanghai, China) were used for detection of primary antibodies and internal control. Signals were imaged with chemiluminescent substrate kit (Thermo Scientific, Rockford, USA) and detected by gel imaging system (Tanon, Shanghai, China), and the ratio of interested proteins to GAPDH was analyzed by Tanon Gis software.

Cells were lysed in RIPA and protein concentration was quantified by BCA Protein Assay Kit (Thermo Fisher Scientific, NCI3225CH). Protocol for western blotting is reported previously^{45 (link)}. Antibodies used are as following: β-catenin (Rb, 1:2000; Abcam), SLUG (Ms,1:1000; Abcam), phosphor-AKT-Ser473 (Rb, 1:1,000; CST, 4060), phosphor-AKT-Thr308 (Rb, 1:1,000; CST, 13038), pan-AKT (Rb, 1:1,000; CST, 4691), GAPDH-HRP (Rb, 1:5,000; Yeasen, 30203ES10), horseradish peroxidase‑conjugated anti‑rabbit IgG (1:2,500; Yeasen, #33101ES60), and anti‑mouse IgG (1:2,500; Yeasen, #33201ES60). Electro‑chemiluminescence method was used to visualize blots (New Cell & Molecular Biotech Co., China, #P10100). GAPDH was used as internal standards. The experiments were replicated three times.

Proteins were lysed from cell samples by RIPA lysis buffer (Cat# P0013B, Beyotime, Shanghai, China) and quantified by a BCA protein concentration assay kit (Cat# PC0020, Solarbio, Beijing, China). Thirty micrograms of protein was loaded, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes for antibody blotting. The primary antibodies were ATG7 (Cat# 67,341–1-Ig) and BECN1 (Cat# 11,306–1-AP), purchased from Proteintech (Wuhan, China). GAPDH (Cat# ab59164, Abcam, Cambridge, MA, USA) was used as a control. Primary antibody incubations were performed at a dilution of 1:2000 in PBS for 16 h at 4 ℃. The secondary antibodies were purchased from Yeasen Biotechnology (Shanghai, China) and included peroxidase-conjugated goat anti-rabbit (1:5000 dilution, Cat# 33101ES60) and anti-mouse IgG (1:5000 dilution, Cat# 33201ES60). Secondary antibody incubations were 1 h at room temperature.