Vdac antibody

After incubation with or without GST-BNIP3, mitochondria were resuspended in 1 ml nondenaturing lysis buffer (1% Triton X-100, 50 mM Tris-Cl, 300 mM NaCl, 5 mM EDTA add 10 mM iodoacetamide, 1 mM PMSF, 2 µg/ml leupetpin, pH 7.4) on ice for 30 min and centrifuged at 12,000 g for 10 min at 4°C. In experiments with VDAC antibody blocking, 0.3 mg/ml anti-VDAC/Porin antibody (Abcam, ab34726) was added to the mitochondrial samples 30 minutes before GST-BNIP3. The supernatants were incubated with a monoclonal BNIP3 antibody (1∶1000, University of Manitoba) or a VDAC antibody (1∶500, Cell Signaling, Danvers, MA) with shaking for 2 hrs on ice. Then, 150 µl Sepharose 4B was added to each sample to incubate for overnight at 4°C with gentle agitation. Immune complexes were precipitated with protein A agarose beads (Invitrogen). Followed by washes in 1 ml nondenaturing lysis buffer for three times, the pellet was resuspended in sample buffer, and proteins were resolved by SDS-PAGE (10% gel) and analyzed by Mass spectrometry and Western blotting with antibodies to BNIP3 (1∶1000) and VDAC (1∶500, Cell Signaling, Danvers, MA), as described previously [5] (link). Western blot bands were quantified using the NIH ImageJ software.

Immediately after culling, mouse tissues were fixed for at least 24 h in 10% neutral buffered formalin (Sigma, HT501128). Tissue sections (4 µm) were cut from paraffin-embedded blocks and stained with hematoxylin (Cell Path, RBA-4201-00A) and eosin (VWR, 341973R), or LC3B antibody (0231-100, Nanotools), or VDAC antibody (Cell Signaling Technology, #4866) as previously described (53 (link)). For the lectin staining, tissue sections were dewaxed in xylene for 5 min followed by rehydration through pure ethanol (2 × 1 min) then 70% ethanol (1 × 1 min). The sections were rinsed in water, then endogenous peroxidase was blocked using Dako Peroxidase block (Agilent, S2023) for 5 min. The sections were rinsed in PBS before the biotinylated lectin was applied at a predetermined optimal dilution (biotinylated AAL and UEA1; 1:2,000) (Vector Labs, B-1395 and B-1065). The lectin was applied for 30 min, then the sections were rinsed in PBS/Tween (2 × 4 min) and had VECTASTAIN Elite ABC-HRP kit (Vector Labs, PK-6100) applied for 30 min as per manufacturer’s instructions. The sections were rinsed in PBS/Tween (2 × 4 min) and then the visualization substrate (3,3′-Diaminobenzidine tetrahydrochloride; Agilent, K3468) was applied for 5 min. This reaction was terminated in water.

Western blottings were principally performed as described in Shabalina et al.^{23 (link)} The levels of mitochondrial respiratory chain proteins (Complex I (NDUFB8), Complex II (SDHB), Complex III (UQCRC2), Complex IV (COX2) and Complex V (α-subunit F_oF₁-ATP-syntase) were examined using the Total OXPHOS Mouse Antibody cocktail from MitoSciences (#MS601, Eugene, OR, USA)) (diluted 1:15 000). UCP1 and voltage-dependent anion channel (VDAC) expressions were examined using either a UCP1 antibody produced in rabbit against the C-terminal UCP1 decapeptide at a dilution of 1:20 000 (in-house product) or a VDAC antibody from Cell Signaling (#4661S) diluted 1:2000. The immunoblottings were visualized in a charge-coupled device camera and expression was quantified using the Image Gauge V3.45 program (Fuji Film Co., Tokyo, Japan).