Ab126575

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.

Frozen SkM was thawed and immediately homogenized in RIPA lysis buffer (Tris-HCl 10 mM, pH 7.4; EDTA 5mM; NaCl 50 mM; sodium deoxycholate 0.1%; triton X-100 1% v/v) supplemented with protease and phosphatase inhibitors (Roche Applied Sciences, Germany) using a motorized pestle system (Pellet Pestle Cordless Drive Unit, DKw Life Sciences, USA). The total lysate was centrifuged at 12,000 rpm three times for 10 min at 4 °C; the supernatant was collected for protein determination using BCA assay (Novagen, Merck Ma, NJ, USA). Protein separation and immunoblotting were performed, as previously described [42 (link)]. Immunoblotting was performed using the following antibodies: Mfn2 (ab50838, Abcam, 1:1000), Mfn1 (ab126575, Abcam, 1:1000), Opa1 (ab42634, Abcam, 1:1000), Drp1 (PA1-16987, Thermo, 1:1000), phosphoserine 616-Drp (5616, Cell Signaling, 1:1000), Total OXPHOS cocktail (ab110413, Abcam, 1:1000), Mic60 (#10179-1-AP, Proteintech, 1:1000), Mic19 (#25625-1-AP, Proteintech, 1:1000), mHsp70 (#MA3-028, Pierce, 1:1000), Oxct1 (#12175-1-AP, Proteintech, 1:1000), Acca2 (#100847, Santa Cruz, 1:1000), Scad (#365953, Santa Cruz, 1:1000), Vlcad (#376239, Santa Cruz, 1:1000), Cpt1 (#393070, Santa Cruz 1:1000), Tom20 (72610, Cell Signaling, 1:1000), and β-Tubulin (T0198, Sigma Chemical Co, 1:5000). Samples of the same muscle were run on the same gels.

The zona pellucida was removed using acidified Tyrode’s solution. Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) with 0.1% Tween 20 and 0.01% Triton X-100 for 10 min at room temperature (RT), permeabilised in 0.55% Triton X-100 in PBS for 15 min at RT, washed 4 times (5 min each) in PBX (PBS with 0.1% Triton X-100) and then blocked in 20% fetal bovine serum in PBS for 1 h at RT. The primary antibodies were used: anti-Mfn2 mouse monoclonal antibody (ab56889, Abcam); anti-Mfn1 mouse monoclonal antibody (ab126575, Abcam); anti- OPA1 rabbit monoclonal (ab157457) and anti-Sox2 rabbit polyclonal (ab97959, Abcam); anti-BnipL3 (ab109414, Abcam); anti-SQSTM1/p62 (sc-28359, Santa Cruz) at dilutions of 1:100 in 20% fetal bovine serum in PBS. Embryos were incubated in primary antibodies solution at 4°C overnight. After that embryos were washed 4 times (5 min each) in PBX and then blocked in 20% fetal bovine serum in PBS for 1 h at RT. The secondary Alexa Fluor-conjugated antibodies (Invitrogen) were used at a dilution of 1:500 in 20% fetal bovine serum in PBS. Embryos were incubated at 4°C for 75 min, then they were washed 4 times (5 min each) in PBX. DNA was visualised using Hoechst 33342 (5 μg/mL; Molecular Probes).