Anti mfn2

Mitochondria-enriched and cytosolic fractions were isolated from cortex, hippocampus, cerebellum and olfactory bulb of WT and Wdfy3^+/lacZ mice as described before^{68 (link)}. Thirty-five µg of proteins were solubilized in SDS sample buffer (Life Technologies, Grand Island, NY) and loaded onto a 4–12% bis-tris gel (Life Technologies) as previously described^{68 (link)}. After transferring proteins with an iBlot apparatus (Life Technologies), membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at 20 °C and subsequently probed overnight at 4 °C with the following antibodies: anti-Lamp2 (Abcam, Cambridge, MA; 1:1,000 dilution), anti-LC3 (Novus Biologicals, Littleton, CO; 1:1,000 dilution), anti-Mfn2 (Proteintech, Rosemont, IL; 1:500 dilution), anti-MnSOD (Millipore, Billerica, MA; 1:1,000 dilution), anti Sqstm1 (Cell Signaling Technology, Danvers, MA; 1:500 dilution), anti-Park2 (Abcam; 1:500 dilution), and anti-Pink1 (Novus Biologicals; 1:1,000 dilution). As a loading control, we used anti-β-actin antibody (Sigma, St. Louis, MO; 1:20,000 dilution, 1 h at 20 °C). Secondary antibodies were from LI-COR (Lincoln, NE; 1:10,000 dilution). Membranes were visualized with the use of the Odyssey Infrared Imaging System (LI-COR) and densitometry analysis carried out with ether the Carestream or ImageJ softwares.

The cells were lysed with lysis buffer (Beyotime, Shanghai China) supplemented with a protease inhibitor solution (Beyotime) and centrifuged at 12 000 g for 10 min at 4 °C. Extracted proteins were separated on 10–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 37 °C, the membranes were incubated overnight with primary antibodies at 4 °C, washed with TBST, and then incubated with secondary antibodies. The western blot results were quantified by densitometric analysis using the Quantity One 4.6.9 software (Bio-Rad).
The following antibodies were used: anti-cleaved caspase-9 (9509T, CST), anti-cleaved caspase-3 (9664T, CST), anti-Bax (#2772, CST), anti-Bcl2 (#3498, CST), anti-cytochrome c (10093, Proteintech), VDAC rabbit mAb (4661, CST), anti-MFN1 (NBP1-71775, Novus Biologicals), DRP1 rabbit mAb (8570, CST), OPA1 rabbit mAb (80471, CST), anti-FIS1 (D122377-0025, BBI life sciences), anti-MFN2 (12186, Proteintech), anti-beta tubulin (10094, Proteintech), anti-PSD95 (20665, Proteintech), spinophilin rabbit mAb (14136, CST, Boston), HP-goat anti-mouse (ZB-2305, Zsbio, Beijing, China), HP-goat anti-rabbit (ZB-2301, Zsbio, Beijing, China), and Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (33112ES60, Yeasen).

TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N‐Acetyl‐L‐cysteine (NAC) were purchased from Sigma‐Aldrich. MitoTracker™ Red FM (M22425), Hoechst 33342 (H1399) and anti‐Ubiquitin WB Antibody (13–1600) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) (1:1,000). Anti‐phospho‐Ubiquitin (Ser65) (ABS1513‐I) was purchased from Merck KGaA company (1:1000). Anti‐P53 (21891–1‐AP), anti‐PINK1 (23274–1‐AP), anti‐Parkin (14060–1‐AP), anti‐Drp1 (12957–1‐AP), anti‐Opa1 (27733–1‐AP), anti‐Mfn1 (13798–1‐AP), anti‐Mfn2 (12186–1‐AP), anti‐Caspase‐9 (10380–1‐AP), anti‐BAX (50599–2‐Ig), anti‐Caspase‐3 (19677–1‐AP), anti‐VDAC1 (10866–1‐AP), anti‐Lamin B (12987–1‐AP), anti‐Cytochrome c (10993–1‐AP), and anti‐β‐actin (60008–1‐Ig) were purchased from ProteinTech Group, Inc., (Chicago, IL, USA) (1:1,000). Anti‐γ‐H2A.X (ab81299) was purchased from Abcam (1:1000). Anti‐AMPKα (2532) and p‐AMPKα (50081) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA) (1:1000). Anti‐phospho‐DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti‐phospho‐DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. (1:1,000).