Anti vdac

Twenty to thirty micrograms of total or mitochondrial protein was separated on SDS-polyacrylamide gel and electro-blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were saturated with blocking buffer for 1 h at room temperature and incubated overnight at 4°C with monoclonal mouse anti-VDAC (1:1000, Abcam), anti-OXPHOS (1:5000, Abcam), anti-IP3R1 (1:1000, Santa Cruz), anti-NDUFA13 (1:1000, Abcam), anti-GAPDH (1:10 000, Abcam) or with polyclonal rabbit anti-MCU (1:500, Abcam), anti-MICU1 (1:500, Thermo Fisher), anti-MICU2 (1:500, Sigma Aldrich), anti-PDH-E1α (1:1000, Abcam), anti-PDHE1α phosphor Ser 293 (1:1000, Abcam), anti-PDHE1α phosphor Ser 300 (1:1000, Millipore), anti-PDHE1α phosphor Ser 232 (1:1000, Calbiochem), anti-PDK4 (1:1000 Novus Biotech), anti-GRP75 (1:1000, Santa Cruz), anti-SIGMA1R (1:1000, Cell Signaling), anti-MFN2 (1:1000, Abcam), and anti-Hsp60 (1:1000, Abcam). Hsp60 and GAPDH were used as loading controls. All immunoblots were developed and quantified using the Odyssey infrared imaging system (LICOR Biosystems) and infrared-labeled secondary antibodies. Band intensities were quantified with ImageJ.

Adriamycin (ADR, catalog number D1515) and dihydroethidium (DHE, catalog number D7008) were obtained from Sigma-Aldrich (St. Louis, MO). Hyperoside (HPS) was purchased Zelang Biological Technology Company (Nanjing, China). Anti-nephrin (catalog number ab58968), anti-podocin (catalog number ab50339), anti-PGC-1α (catalog number ab54481), phosphor Anti-Drp1 (S637) (catalog number ab193216) and anti-VDAC (voltage-dependent anion channel, catalog number ab34726) antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-Drp1 (catalog number sc-32898) and anti-Mfn-1 (catalog number sc-166644) antibodies were purchased from Santa Cruz (Santa Cruz, CA). anti-GAPDH was purchased from Sanying biotechnology (Wuhan, China, catalog number 10494-1-AP). All secondary antibodies for immunoblot analysis were from Zhongshan Golden Bridge Biotechnology (Beijing, China, catalog number ZB-2301). MitoSOX (catalog number M36008) and 2’,7’-dichlorofluorescein diacetate (DCFDA, catalog number C6827) were from Invitrogen (Carlsbad, CA, USA).

Liver mitochondria were isolated in a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris–HCl, 1 mM EDTA, and 5 mM EGTA, pH 7.4, at 4 °C according to [16 (link)]. In addition, 10 μg of mitochondrial proteins were used for Western immunoblotting analysis. Anti-TFAM (1:50,000), anti-VDAC (1:50,000, Abcam, Cambridge, UK), anti-OGG1 (1:2500, Abcam, Cambridge, UK), anti-APE1 (1:5000, Abcam, Cambridge, UK), anti-MFN2 (1:5000, Abnova, Taipei, Taiwan), anti-DRP1 (1:2500, Abnova, Taipei, Taiwan), anti-Cyt c (1:500, Pharmingen, San Diego, CA, USA), and anti-Lon Protease (1:10,000) were used as primary antibodies. The antibodies against TFAM and Lon were custom-made and kindly donated, respectively, by Dr. H. Hinagaki (Department of Chemistry, National Industrial Research Institute of Nagoya, Nagoya-shi, Aichi, Japan) and Dr. C. Suzuki (Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA). The proteins were detected by chemiluminescence and immunoreactive bands were quantified using the Image Lab Software (BioRad Laboratories Inc., Hercules, CA, USA) and normalized against VDAC-expression.

RIPA buffer with protease inhibitors was added to brain tissue weighing 6–7 mg and brain was homogenized. The obtained samples were centrifuged at 10,000× g for 20 min and then the pellet was solubilized with Laemmle buffer. An aliquot of the non-synaptic mitochondria, suspension from synaptosomal fraction, and suspension from myelin fraction were solubilized with Laemmle buffer and heated at 95 °C for 3 min. The proteins from brain tissue and brain mitochondria were separated by electrophoresis (12.5% SDS-PAGE). Then, the proteins were transferred from the gel onto a nitrocellulose membrane (0.2 µm). The membrane was stained with appropriate antibodies. The monoclonal antibody to CNPase was as described previously in [53 (link)], and the monoclonal antibody to ADAP1 was as described previously in [54 (link)]. The polyclonal anti-ANT1, anti-ANT2, and anti-VDAC, as well as the monoclonal anti-CyP-D antibody, were from Abcam (Cambridge, UK). Tom20 (Cell Signaling, Danvers, MA, USA) and myelin basic protein (MBP) (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for normalization of proteins.

OSCC cell lines (HSC-3 and HN12) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. The cells were routinely cultured in high glucose DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, United States) and 1% antibiotics at 37°C in a 5% CO₂ incubator. The primary antibodies anti-PERK, anti-p-PERK, anti-eIF2α, anti-p-eIF2α were purchased from Cell Signaling Technology (1:1000, United States). The primary antibodies anti-ATF4, anti-caspase-3, anti-cleaved-caspase-3, anti-Bcl2, anti-Bax were obtained from Affinity (1:500, United States). Antibodies α-tubulin, anti-HSP60, anti-IF1, anti-VDAC, and anti-CHOP were obtained from Abcam (1:1000, Cambridge, MA). The OXPHOS complexes were obtained from Thermo Fisher Scientific (1:1000, United States). Antibody p-Ser was obtained from Abcam (1:1000, AP0932, China). Goat anti-rabbit/mouse secondary antibody was purchased from ZSGB-BIO (Beijing, China). Nebivolol was purchased from Selleck (Shanghai, China). 4-Phenylbutyrate (4-PBA) was purchased from Sigma-Aldrich (St. Louis, MO, United States). MitoTracker Red was obtained from Solarbio (Beijing, China).

Whole cell or mitochondrial lysates from differentiated C2C12 cells were prepared with RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 0.5 mM EDTA, 150 mM sodium chloride) supplemented with protease inhibitor cocktail (Roche). Forty μg of lysate, 20 μg of mitochondrial pellet, or 10 μg of supernatant were run on a 10% gel (Bio-Rad) and transferred to PVDF membrane (Immobilon). The membrane was probed with rabbit polyclonal anti-NMNAT1 (1:500 dilution) as previously described (Zhang et al., 2009 (link)) or anti-VDAC (Abcam) followed by secondary antibody incubation. Immunoblots were developed using SuperSignal West femto kit (Thermo Fisher Scientific) on a Bio-Rad imaging system. Blots were then stripped and re-probed with HRP-conjugated β-actin antibody (Abcam).

Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail. Protein concentrations were determined by the BCA protein assay. Total proteins (40 μg) were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore, Massachusetts, USA). After blocking with 5% BSA in TBST, the membranes were incubated with the following primary antibodies at 4 °C overnight; anti-CD9, anti-CD63, anti-TSG101, anti-VDAC, anti-LAMP1 were obtained from Abcam (Abcam, USA); and anti-p38/p-p38, anti-ERK/p-ERK, anti-JNK/p-JNK, anti-p65/p-p65, anti-IKKα/p-IKKα, anti-Iκbα/p-Iκbα, anti-Sirt1, anti-GAPDH, anti-β-actin were obtained from Cell Signaling Technology (CST, USA); and anti-PGC1α, anti-cox15, anti-NDUFV2, anti-ATP5D, anti-ATP5H were obtained from Proteintech Biotechnology (Proteintech, USA); and anti-TFAM was obtained from Absin Bioscience (Absin, Shanghai, China); and anti-TOM20, anti-CALR were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Thereafter, the membranes were incubated with HRP-conjugated anti-mouse and anti-rabbit IgG (1:4000, CST, USA), respectively, at room temperature for 1 h. ECL reagent was used to develop the membrane and signal was detected on a digital image system (Bio-Rad, USA).