Anti manf

Total protein was extracted from mouse liver tissues or HepG2 cells with RIPA lysis buffer (Beyotime) containing phosphatase inhibitor cocktail A (Beyotime) and protease inhibitor (Roche, Basel, Switzerland), and the protein concentration was measured with a BCA Protein Assay kit (Beyotime). Protein samples (30 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (120 mL/L gel) and transferred to a 0.22 μm polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). Then, the membrane was blocked with 50 g/L BSA for 2 h at room temperature, followed by incubation with primary antibody at 4 °C for 12-16 h and then secondary antibody for 1-2 h at room temperature. The protein expression level was detected using the chemiluminescence method, and the images were captured by a computer. GAPDH served as a control. The antibodies included anti-MANF (Abcam, Cambridge, MA, United States, ab67271, 1:1000), anti-GAPDH (Proteintech Group, Wuhan, Hubei, China, 60004-1-Ig, 1:5000) and HRP-coupled goat anti-rabbit or goat anti-mouse IgG secondary antibody (Zhongshan Co., Beijing, China, ZB-2301, ZB-2305, 1:5000).