Mic60

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.