Anti hsp60

Anti‐MITOL rabbit polyclonal antibody was produced as described previously. Anti‐β‐actin and anti‐FLAG‐M2 antibodies were purchased from Sigma. Anti‐Parkin and anti‐VDAC were purchased from Cell Signaling Technology. Anti‐Parkin (phospho‐Ser65) was purchased from UBIQUIGENT. Anti‐Tom20 and anti‐calnexin were purchased from Santa Cruz Biotechnology. Anti‐ubiquitin and anti‐GFP were purchased from MBL. Anti‐myc was obtained from Recenttec. Anti‐HA was obtained from Proteintech. Anti‐GST and BAPTA‐AM were purchased from Wako. Anti‐HSP60 was purchased from Enzo Life Sciences. Anti‐FKBP38 and anti‐K63‐chain were purchased from Abcam. Anti‐K48‐chain was purchased from Millipore. Carbonyl cyanide m‐chlorophenylhydrazone (CCCP) and chloroquine diphosphate were obtained from Wako. MG132, lactacystin, Z‐VAD‐FMK, and bafilomycin A1 were obtained from Peptide Institute. Cycloheximide (CHX) was purchased from Sigma.

For detecting oligomeric Drp1, cultured cortical neurons were collected in lysis buffer (100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 50 mM Tris-Cl, protease and phosphatase inhibitor cocktails, pH 7.4). Then, non-reducing SDS-PAGE was performed using samples that were either left untreated or treated with DTT (1 μM). For mitochondrial fractionation, cultured cortical neurons were rinsed with PBS and collected in pre-chilled mitochondria buffer (250 mM sucrose, 10 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES, protease and phosphatase inhibitor cocktails, pH 7.5, 4 °C). Supernatant was obtained by centrifugation at 500 × g for 5 min and 10 000 × g for 15 min at 4 °C. The pellet and supernatant represented the mitochondrial and cytosolic fractions, respectively. Immunoblotting was performed using the following antibodies: anti-Drp1 (1:2000), anti-pDrp1^S616 (1:500), anti-CDK5 (1:1000), anti-p35 (1:1000), anti-β-Actin (Sigma-Aldrich, #A5441; 1:2000), anti-HSP60 (Enzo Life Sciences, Farmingdale, NY, USA, #ADI-SPA-806; 1:1000), anti-GAPDH (Santa Cruz Biotechnology, #6C5; 1:1000), anti-Mfn2 (Abcam, Cambridge, MA, USA, #ab56889; 1:1000), anti-Opa1 (BD Biosciences, #612606; 1:1000), anti-Mff (Sigma-Aldrich, #HPA010968; 1:1000) and anti-Fis1 (Abcam, #ab96764; 1:1000).