Anti cytochrome c

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).

The radio immunoprecipitation assay (RIPA) strong lysate (Beyotime Biotechnology, Shanghai, China, P0013B) was used to lyse the spleen tissues for 30 min, after which they were centrifuged at 12,000× g for 15 min at 4 °C. The quantification of protein concentrations was performed using a BCA protein concentration detection kit (Beyotime Biotechnology, Shanghai, China, P0010). The tissue proteins were first incubated to primary antibodies at 4 °C overnight, and then to matching secondary antibodies for 1 h, and finally they were incubated using the LumiBest ECL substrate solution kit for visualization (ShareBio, Shanghai, China, SB-WB011). The protein band intensity was measured using Image-J software.
Antibodies used in this study: anti-TNF-α (Immunoway, Suzhou, China, YT4689, 1:1000), anti-IL-1β (Beyotime Biotechnology, Shanghai, China, AF7209), anti-NF-κB p65 (ABclone, Wuhan, China, A2547, 1:1000), anti-IFN-γ (Immunoway, Suzhou, China, YT2279, 1:1000), anti-Caspase-3 (Proteintech, Wuhan, China, 19677-1-AP, 1:1000) anti-Bcl-2 (ABclone, China, A19693, 1:1000), anti-Bax (Proteintech, China, 50599-2-lg, 1:1000), anti-Cytochrome C (Proteintech, China, 10993-1-AP, 1:1000), anti-β-actin (MultiSciences, Hangzhou, China, #HA-R1207-1-200, 1:1000), HRP Goat Anti-Rabbit IgG (H + L) (Abclone, China, #AS014, 1:4000), and HRP Goat Anti-Mouse IgG (H + L) (Abclone, China, #AS003, 1:4000).

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).