Ab137207

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.

Cells were grown in 60-mm petri dishes. Cells were washed with PBS (0.01 M phosphate, 0.0027 M KCl, 0.14 M NaCl, pH 7.4), then 0.1 mL of TET lysis Buffer (0.02 M Tris, 0.005 M EDTA, 1% Triton-X, pH= 7.4) was added to cells on ice and cells were collected. The lysate was incubated for 45 min on ice and vortexed 3 times. Proteins were separated on a gradient SDS-PAGE (Mini-PROTEAN TGX Gels, BIO-RAD, Hercules, CA, USA ) and the proteins were transferred to a PVDF membrane (88518, Thermo Fisher, Waltham, MA, USA ). The membrane was blocked with 5% Bovine Serum Albumin (BSA, A9647-100G, Sigma-Aldrich, St. Luis, MO, USA). The membrane was incubated with anti-SGLT1 (anti-SLC5A1 TA324226 from Origene, Rockville, MD, USA), anti-SGLT2 (ab137207, Abcam, Cambridge, UK), or anti-GAPDH (ABS16, EMD Millipore, Rockville, MD, USA) in 1% BSA solution. Super Signal West Femto and Pico Maximum Sensitivity Substrates (Sigma-Aldrich, St. Luis, MO, USA) were used for chemiluminescence signals. The results were quantitated using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). The data were analysed using Image Lab software from Bio-Rad (Hercules, CA, USA).