Ab110412

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).

Antibodies anti‐COX4, anti‐UQCRC2, anti‐NDUFA9, anti‐ATP5A, anti‐SDHA were from Abcam (OXPHOS cocktail ab110412, dilution 1:2,000); anti‐SDHB (ab14714, dilution 1:200) and anti‐COX1 (ab14705, dilution 1:2,000) were from Abcam; anti‐HSC70 was from Santa Cruz (sc‐7298, dilution 1:1,000); anti‐GAPDH was from Abcam (ab8245, dilution 1:40,000); anti‐P62 was from Abnova (H00008878‐M01, dilution 1:1,000); anti‐LC3‐I/II was from Novus Biologicals (NB100‐2220, dilution 1:1,000); anti‐LAMP1 was from Cell Signaling (3243, dilution 1:1,000); anti‐PINK1 was from Novus (BC100‐494, dilution 1:1,000); and anti‐Parkin was from Abcam (ab77924, dilution 1:500).

Western blots were performed using protein extracts remaining from the enzymatic assay preparations. Protein concentration was determined by a bicinchoninic acid (BCA) assay (Pierce #23227) and samples were prepared with 4x LDS sample buffer (Novex #NP007). 30 μg protein per sample were resolved by standard western blot procedure on 4–12% bis–tris protein gels (Novex #WG1403BX10), and then transferred to PVDF membranes using the semi-dry system from Life Technologies/Invitrogen. Membranes were cut based on molecular ladder size to detect proteins of different size from the same membrane with different antibodies. Detection was achieved using an OXPHOS antibody cocktail (Abcam #ab110412, 1:1000), or antibodies against Porin1 (Abcam #ab15895, 1:1000), citrate synthase (Abcam #ab96600, 1:1000), CD38 (R&D System #MAB24041, 2 μg ml⁻¹), GAPDH (Abcam #ab37168, 1:5000) and HSC70 (Santa Cruz, sc-7298; 1:50000), with the relevant secondary antibodies and using enhanced chemiluminescence (ECL; Pierce #321016) detected by standard autoradiography with exposure time adjusted to the expression of each protein. Complexes I–III and V were correctly detected in all samples but complex IV was only detected in less than 20% of samples and was excluded from the analysis. Films were scanned and quantified using Image J.