Ab14713

Immunoblotting was performed using 4–12% gradient gels through MOPS SDS-PAGE, as previously described [54 (link), 55 (link), 57 (link)–60 (link)]. Normalization of protein content was assessed using the Bradford Method [56 (link)]. Primary antibodies utilized in the study included the following: total OXPHOS Blue Native WV Antibody Cocktail (ab110412) (anti-NDUFA9 (complex I, ab14713, Abcam, Cambridge, MA), anti-SDHA (complex II, ab14715, Abcam), anti-UQCRC2 (complex III, ab14745, Abcam), anti-COX IV (complex IV, ab14744, Abcam), and anti-ATP5A (complex V/ATP Synthase, ab14748, Abcam)), anti-ATP5F1 (complex V/ATP Synthase, ab117991, Abcam), and anti-VDAC (#4866, Cell Signaling Technology, Danvers, MA). Goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugate 1:10,000 (Thermofisher Scientific) and goat anti-rabbit IgG HRP conjugate 1:5000 (Abcam) were used as the secondary antibodies. Normalization of protein content was through VDAC expression. Chemiluminescence quantified with Radiance Chemiluminescent Substrate (Azure Biosystems, Dublin, CA), per manufacturer’s instructions and imaged using the G:Box Bioimaging system (Syngene, Frederick, MD). GeneSnap/GeneTools software (Syngene) was used to acquire images. Densitometry was analyzed using Fiji Software (NIH, Bethesda, MD).

PC9 and PC9M2 cells were harvested and lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM EDTA, 50 mM Tris, pH 7.4) in the presence of complete protease inhibitor mixture (Roche). The extracted proteins were separated using 4–12% Tris-MOPS gel (NuPAGE, Invitrogen) and transferred onto polyvinylidene difluoride membrane (PVDF, BioRad). The membranes were probed using the following antibodies: monoclonal anti-MFN1 (1:1000, Abcam, ab57602), monoclonal anti-MFN2 (1:1000, Abnova, H00009927-M03), monoclonal anti-Opa1 (1:1000, BD Biosciences, 612607), monoclonal anti-DLP1 (1:1000, BD Biosciences, 611112), monoclonal anti-β-catenin (1:1000, BD Biosciences, 610154), rabbit polyclonal anti-OMA1 (1:1000, Proteintech, 17116-1-AP), rabbit polyclonal anti-YME1L (1:1000, Proteintech, 11510-1-AP), monoclonal anti-NDUFA9 (1:1000, Abcam, ab14713), monoclonal anti-SDHA (1:1000, Abcam, ab14715), and monoclonal anti-β-actin (1:100000, Sigma, A5316). Isotype-matched, horseradish peroxidase-conjugated secondary antibodies (Amersham) were used, followed by detection using chemiluminescence (Amersham).

Cells were lysed using a buffer with sodium dodecyl sulfate (SDS) (Cell Signaling Technology, Danvers, MA, USA, #9803; following the supplier’s protocol), followed by centrifugation at 12,000× g for 10 min, and the supernatants were recovered as whole-cell extracts. Protein content was measured using the Bradford reagent. Protein extract samples were loaded on 12% polyacrylamide gels (Invitrogen #NP0343BOX, supplied by Fisher Scientific Co., Boston, MA, USA) and electrophoresed for 2 h at 14.5 V/cm. Wet electroblotting transfer was performed for 3 h on ice onto a polyvinylidene difluoride (PVDF) membrane. The primary antibodies included anti-Ape1 (Novus Inc. Guaynabo, PR, USA, #NB100-101; used at 1:1000), anti-DNA2 (Abcam Inc., Waltham, MA, USA, #ab96488; used at 1:500), anti-ExoG (Abcam #ab77736; used at 1:500), anti-Fen1 (Novus NB100-320; used at 1:700), anti-NDUFA9 (specific for a Complex I protein; Abcam ab14713; used at 1:1000), and anti-TFAM (Cell Signaling 7495; used at 1:1000). The secondary antibodies (both used at 1:10,000) were goat anti-rabbit IgG (Licor Inc., Omaha, NB, USA, #925-68021) and goat anti-mouse IgG (Licor 926-32210). The developed blots were placed in distilled water, scanned on a Li-COR Odyssey (Licor Inc. Omaha, NB, USA), and quantified using Image Studio software (version 5.5.4), with n ≥ 3.

Mitochondria were isolated using Dounce or Balch homogenizers, followed by standard differential centrifugation^{13 (link),50 (link),70 (link)}. Experimental procedure and antibodies for NBGE were used as described previously^{57 (link),71 (link)}. In brief, digitonin-solubilised mitochondria were separated on NativePAGE Novex Bis-Tris 3–12% gradient gels. After electrophoresis, the gels were incubated in transfer buffer containing 0.1% SDS for 10 min and proteins were transferred to PVDF membranes probed with specific antibodies (all diluted 1:500, except for VDAC1 and HSP60 diluted 1:1000) against complex I (NDUFA9, ab14713, Abcam; or NDUFB8, ab110242, Abcam), CII (SDHA, 14715, Abcam), CIII (Core2, ab14745, Abcam), CIV (COXVa, ab110262, Abcam) and CV (ATP5A, ab14748, Abcam; or ATP5B, HPA001520, Sigma Aldrich), and VDAC1 (ab15895, Abcam) or HSP60 (12165S, Cell Signaling) as the loading control.