Detailed Western Blot Methodology

Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).