Any kd mini protean

E. coli cells were lysed by sonication in 20 mM Tris buffer (pH 7.5) buffer A supplemented with protease inhibitor cocktail and DNase (Sigma, St. Louis, MO) and 200 mM NaCl. The cell lysate was clarified by centrifugation at 25,000 × g, and the supernatant was loaded onto a HisTrap HP column (GE healthcare, Marlborough, MA). The His₆-tagged FRP was eluted with buffer A containing 300 mM imidazole and further purified by gel filtration chromatography by using a HiPrep Sephacryl S-200 HR (GE healthcare, Marlborough, MA) column and an isocratic flow of buffer A. The purity of FRP was confirmed by SDS–PAGE by using a precast gradient gel (Any kD™ Mini-PROTEAN, Bio-Rad, CA). The concentration of FRP was determined by 280 nm absorption on a NanoDrop spectrophotometer (Thermo Scientific, MA, USA).

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

MDA-MB-231 cells were seeded on top of either compliant (1kPa) or stiff (10kPa) gels for 24 hours and treated with or without the FAK inhibitor PF573228 (MilliporeSigma). Cells were rinsed with 1X PBS and lysed with 4X SDS sample buffer (4X Tris-Cl/SDS, pH6.8, 30% v/v glycerol, 10% w/v SDS, 0.09% v/v 2-mercaptoethanol, and 0.012% w/v Bromophenol Blue). Standard SDS-PAGE was conducted usingBio-Rad Any kD Mini-PROTEAN (4569035; Bio-Rad gels and PVDF membranes (Bio-rad). Membrane washing steps were performed with 0.1% polyoxyethylene 20 sorbitan monolaurate (Tween; JT Baker, Phillipsburg, NJ) in Tris-buffered saline. Blocking was performed with 5% milk in the washing buffer. Primary antibodies (GAPDH Biolegend poly6314; CSF-1 Santa Cruz sc-365779) were diluted in blocking buffer at 1:1000 dilution and applied to the membranes overnight at 4°C. Horseradish-peroxidase conjugated secondary antibodies were applied to the membranes in blocking buffer at 1:2000 dilution for 1 hour at room temperature. Membranes were imaged using SuperSignal chemiluminescent substrate and a FujiFilm ImageQuant LAS-4000. Quantification of protein expression was normalized to GAPDH loading control and densitometry was performed using Fiji.