Anti mfn1

Primary hippocampal neurons were lysed in lysis buffer (10 mM Tris at pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 1% Triton X-100, 1% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich) as previously described [40] (link). The extracted proteins were diluted into indicated amounts and separated on 8–12.5% SDS/PAGE and transferred onto polyvinylidene difluoride (PVDF) blotting membranes (BioRad). The membranes were blocked in 5% non-fat milk (Cell Signaling) dissolved in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 for 1 h at room temperature and probed with different antibodies: anti-DLP1 (1:3000, BD Biosciences), anti-phospho-Drp1 (Ser 616) (1:1000, Cell Signaling), anti-OPA1 (1:1000, BD Biosciences), anti-Mfn1 (1:1000, Novus Biologicals), anti-Mfn2 (1:1000, Sigma-Aldrich), anti-VDAC (1:1000, Cell Signaling), anti-cleaved-caspase-3 (Asp175) (1:1000, Cell Signaling), and anti-β-actin (1:10,000, Sigma-Aldrich). ECL (GE Healthcare) and Western Bright ECL (Advansta) were used to develop the film. Quantification analysis was performed on scanned films using Image J (NIH).