Glycolysis antibody sampler kit

Cell lysates were prepared with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin. 1 mM PMSF was added immediately prior to use. The protein concentration was measured by DC protein assay (Bio-Rad). The primary antibodies and dilutions were used as follow: glycolysis antibody sampler kits (#8337&12866, Cell Signalling) used at 1:1000; the OXPHOS human WB Antibody cocktail, anti-CS and IDH2 used at 1:1000 (Abcam); anti-HSP60 and SUCLA2 (Santa Cruz); anti phospho AMPK T172 and AMPK, anti phospho ribosomal S6 Ser235/236 and S6; anti phospho mTOR Ser2481 and mTOR used at 1:1000 (Cell Signaling). Generally, 20 μg of protein lysate were loaded on SDS-PAGE gel. Immunoblotting quantification was carried out on an Odyssey Imager (Licor).

Cell lysates were prepared with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin. 1 mM PMSF was added immediately prior to use. The protein concentration was measured by DC protein assay (Bio-Rad, Irvine, CA). Quick neuron nuclear extract preparation: the cells were rinsed with PBS once, and plates placed on ice and 1 ml of ice cold Buffer A (25 mM Hepes pH 7.0, 25 mM KCl, 0.05 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.1% NP-40, 1 mM DTT) added. The plate was scraped and cells were transferred into an Eppendorf tube, which was centrifuged and rinsed once with Buffer A (no NP-40). The pellet was resuspended in PBS, and 2x SDS PAGE sample buffer added, prior to boiling for 10 min at 95°C. The following primary antibodies and dilutions were used: antibodies against the major glycolysis enzymes were from Cell Signaling sold as glycolysis antibody sampler kits (#8337&12866); all were used at 1:1000; Rabbit anti-TFAM (Cell Signaling) used at 1:1000; Rabbit anti-CS, IDH2, PGC-1α and ATP5O (Abcam, Cambridge, United Kingdom) used at 1:1000, and mouse anti-Sucla2 and goat anti-Hsp60 (Santa Cruz Biotechnology) used at 1:1000. Immunoblotting results were analyzed by Odyssey Imager (Licor, Lincoln, NE) scanning.

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).