Protein Extraction and Western Blot Analysis

Protein samples were extracted from cells or tissues with RIPA lysis buffer (Beyotime, P0013B, Shanghai, CN). Protein concentration was detected with a BCA Protein Assay Kit (Beyotime, P0012S, Shanghai, CN). Western blotting was prepared according to previous study[27 (link)] Primary antibodies against GAPDH (1:1000, AB-P-R 001, Hangzhou Xianzhi Biological Co., Ltd., Hangzhou, CN), pepsin (1:1000, DF8591, Affinity Biosciences, Melbourne, AUS), Bax (1:1000, 50599-2-Ig, San Ying Biotechnology, Wuhan, CN), Bcl-2 (1:1000, 26593-1-AP, San Ying Biotechnology, Wuhan, CN), caspase3 (1:1000, Ab184787, Abcam, Cambridge, UK), GLUT1 (1:1000, AF6731, Affinity Biosciences,Melbourne, AUS), MCT4 (1:1000, DF4182, Affinity Biosciences, Melbourne, AUS), and HK-II (1:1000, DF6176, Affinity Biosciences, Melbourne, AUS) were used to detect protein levels of these molecules. The next day, the membrane was soaked with secondary antibodies [HRP-labeled sheep anti-rabbit secondary antibody, 1:10000, BA1054, Wuhan Bode Bioengineering Co., Ltd., Wuhan, CN; goat anti-mouse IgG (H + L) HRP, 1:5000, S0002, Affinity Biosciences, Melbourne, AUS]. The membrane was subsequently scanned (Canon, K10486, Jap), and grayscale value of protein expression was analyzed by BandScan (Glyko, USA).