Anti dld

Frozen-thawed mitochondrial pellets were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to a methanol-activated polyvinylidene difluoride membrane. Immunoblotting was performed as recommended by the manufacturers of the antibodies. Mouse monoclonal anti-cyclophilin D (cypD; Mitosciences, Eugene, OR, USA), rabbit polyclonals anti-OGDH, anti-DLST, anti-DLD, anti-VDAC1, anti-CypD, anti-GR, anti-GPX1, anti-TRX, and anti-PRX3 (Abcam, Cambridge, UK) primary antibodies were used at concentrations of 1 microg/mL, while rabbit polyclonal anti-manganese superoxide dismutase (MnSOD; Abcam) at 0.2 microg/mL. Immunoreactivity was detected using the appropriate peroxidase-linked secondary antibody (in 1:4000 dilution, donkey anti-mouse or donkey anti-rabbit; Jackson Immunochemicals Europe Ltd., Cambridgeshire, UK) and enhanced chemiluminescence detection reagent (ECL system; Amersham Biosciences GE Healthcare Europe GmbH, Vienna, Austria).

A Cell Counting Kit-8 (CCK-8, Cat# CK04) was purchased from Dojindo (Kumamoto, Japan). Pooled antisense oligonucleotides (i.e., siRNAs) against human OVOL2, P65, and GLUT1 were purchased from GenePharma (Shanghai, China). Antibodies against OVOL2 (Cat# ab101580) and P65 (Cat# ab16502) were purchased from Abcam (Cambridge, MA, USA). The following antibodies were used: Glycolysis Antibody Sampler Kits (Cell Signaling Technology; Cat# 12866 and Cat# 8337), anti-GLUT1, anti-GLUT2, anti-GLUT3, anti-GLUT4, anti-PGK1, anti-GCK, anti-PDP2, anti-DLD, anti-PCK1, anti-SDHA, anti-G6PD (all from Abcam; Cat# ab115730, ab234440, ab191071, ab188317, ab38007, ab184169, ab99170, ab133551, ab28455, ab14715, and ab210702, respectively), anti-GAPDH anti-Lamin B, anti-actin and anti-Calnexin (all from Cell Signaling Technology; Cat# 8884, 13435, 3700 and 2433, respectively).

Acetonitrile, trifluoroacetic acid (TFA), K₃Fe(CN)₆, HCCA, 5-hydroxydecanote (5-HD), Nycodenz and urea were purchased from Sigma (St. Louis, MO, USA). Bromophenol blue, acrylamide, CHAPS, immobilized pH gradient (IPG) strips, two-dimensional polyacrylamide gel electrophoresis (2DE) Quant kit, 2DE Clean-up Kit, polyvinylidene fluoride membrane and low melting point agarose were purchased from Bio-Rad (Hercules, CA, USA). Glycerol, glycine, ammonium persulfate, N, N′-methylene acrylamide, SDS, Tris, EDTA, sucrose and mannitol were supplied by Amresco (Solon, OH, USA). Secondary antibodies and anti-GRP75, anti-NDUFV1, anti-DLD and anti-cytochrome oxidase IV (COX IV) primary antibodies were obtained from Abcam (Cambridge, UK). TEMED was from Amersham (UK). Butanol was from Merck (Darmstadt, Germany). Trypsin was from Promega (Madison, WI, USA). Pentobarbital sodium and Tween 20 were both from Solarbio (Beijing, China). All other unlisted reagents were of analytical grade or guaranteed reagents.