Ab137773

Western blots were performed using 4% to 20% Mini-Protein TGX Protein gels (BioRad). Primary antibodies used were: rabbit anti-OGDH (1:1000; ab137773, Abcam), Rabbit anti-DLST (1:1000; HPA003010, Sigma), mouse anti-ATP5A (1:2000; ab14748, Abcam), and mouse-anti-actin (clone:C4) (1:10 000; 8691002, MP Biomedicals). For quantification of proteins expressed from the pcDNA3.1-OGDH (WT or N320S) in HEK293 cells, we used a capillary electrophoresis-based protein analysis system (WES; ProteinSImple, San Jose, California). Cell extracts (0.1 mg/mL) were separated and visualized using the standard instrumental protocol. Primary antibodies used were: rabbit anti-OGDH (1:10, ab137773, Abcam), and rabbit anti-GFP (1:500, A-11122, ThermoFisher).

For each sample, 5 μg protein (in mitochondrial lysate) was loaded into an Any kD™ Mini-PROTEAN® (Bio-rad) acrylamide gel. The primary antibodies used for probing each enzyme were anti-Cs (ab96600, Abcam, 1:5000), anti-Idh2 (ab94359, Abcam, 1:500), anti-Ogdh (ab137773, Abcam, 1:5000), anti-Suclg2 (ab187996, Abcam, 1:5000), anti-Sdha1 (ab139181, Abcam, 1:2000), anti-Fh (ab95947, Abcam, 1:2000), anti-Mdh2 (ab96193, Abcam, 1:1000), anti-Pcca (A304–324A, Bethyl, 1:5000), anti-Vdac1 (ab15895, Abcam, 1:2000). Anti-Pccb (1:4000) and anti-Mut (1:5000), were made by Bio-Synthesis, Lewisville, TX, USA against peptides containing amino acids NH2-CRLRATFAGLYSSLDVGEEGDQ-OH for Pccb and NH2-CPEWAALAKKQLKGKNPED-OH for Mut. Chicken anti-Rabbit IgG-HRP (sc-2955, Santa Cruz, 1:2000) was used as secondary antibody. Protein quantification was performed by measuring the amount of absorbance on a ChemiDoc™ MP Imaging System (170–8280; Bio-Rad) using Image Lab Software 5.1 by BioRad.
All immunoblot normalizations were performed using Vdac1, a mitochondrial membrane voltage dependent anion channel which is independent of the propionate and TCA cycle pathways, as loading control