Af6731

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).

Hippocampal tissues or cells were homogenized in buffer (0.5 mol/L Tris; 1% NP40; 1% Triton X-100; 1 g/L sodium dodecyl sulfate; 1.5 mol/L NaCl; 0.2 mol/L EDTA; 0.01 mol/L EGTA; and protease inhibitor and/or phosphatase inhibitor), sonicated, and incubated at −20°C for 20 minutes, followed by centrifugation at 12,000 g for 20 minutes at 4°C. The supernatant was collected and protein concentration determined by the bicinchoninic acid (BCA) method. Next, 50 μg protein were loaded on 10% SDS polyacrylamide gel. The primary antibodies used were anti-TRPC6 (Alomone, ACC-017), anti-PSD 95 (Abcam, ab238135), anti-SNAP25(Abcam, ab109105), anti-SYP (Abcam, ab32127), anti-MFN1 (Abcam, ab126575), anti-MFN2 (Abcam, ab124773), anti-Drp1(Abcam, ab184247), anti-p-Drp1(mice Ser 622; CST, 3455), anti-p-Drp1 (mice Ser 643; Abcam, ab193216), anti-GLUT1-5 antibody (Affinity, AF6731, DF7510, AF5463, AF5386, DF13545), anti-SGLT1 (Invitrogen, PA5-77460), and anti-SGLT2 (Abcam, ab137207), followed by incubation with the secondary antibodies (ZSGB-BIO). Protein expression was normalized to GAPDH intensity or total protein content. See complete unedited blots in the supplemental material.