Mouse immunoglobulin g igg

Dexamethasone, 3-isobutylmethylxanthine, insulin, sodium fluoride, sodium orthovanadate, fetal bovine serum (Australian origin), benzamidine, and mouse immunoglobulin G (IgG) were purchased from Sigma. LB base, ampicillin, kanamycin, aprotinin, leupeptin, and pepstatin A were obtained from American Bioanalytical (Natick, MA). Calf serum was purchased from Life Science, and Dulbecco’s modified Eagle’s medium (DMEM) was from Mediatech (Herndon, VA). Transfection reagent and the pcDNA 3.1 expression vector were purchased from Life Science. A BCA protein assay kit was from Pierce. Protein A or G magnetic beads was from Santa Cruz Biotechnology (Santa Cruz, CA). Penicillin, streptomycin, and trypsin were purchased from Life Science. Fatty acid, Lactate, Glucose, ROS, GSSG, and GSH were measured by using commercial available kits.

Dexamethasone, 3-isobutylmethylxanthine, insulin, sodium fluoride, sodium orthovanadate, fetal bovine serum (Australian origin), benzamidine, and mouse immunoglobulin G (IgG) were purchased from Sigma (St. Louis, MO). LB base, ampicillin, kanamycin, aprotinin, leupeptin, and pepstatin A were obtained from American Bioanalytical (Natick, MA). Calf serum was purchased from Life Science (Cambridge, MA), and Dulbecco’s modified Eagle’s medium (DMEM) was from Mediatech (Herndon, VA). Transfection reagent and the pcDNA 3.1 expression vector were purchased from Life Science. A BCA protein assay kit was from Pierce. Protein A or G magnetic beads was from Santa Cruz Biotechnology (Santa Cruz, CA). Penicillin, streptomycin, and trypsin were purchased from Life Science.

For co-immunoprecipitation (co-IP), cell lysates were prepared by adding lysis buffer (150 mM NaCl, 1% IGEPAL® CA-630, 50 mM Tris·Cl; pH 8.0) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysate was immunoprecipitated using 2–3 μg of antibody (specificity indicated in the figures), mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO, USA), and incubated with 50 μL of Protein G-Sepharose (GE Healthcare, Chicago, IL, USA). The immunoprecipitates were washed three times in 1 mL of ice-cold lysis buffer, followed by additional wash an additional time with 1 mL of 50 mM Tris·Cl (pH 8.0). The precipitated proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8%–12%). For western blot analysis, the blots were incubated using the antibody indicated in the figures. All co-IPs and western blot analyses were performed more than twice to confirm that the data were reproducible. The following antibodies were used in the co-IPs and western blot analyses: monoclonal anti-FLAG antibody (1:2000, Clone M2; Sigma-Aldrich), monoclonal anti-HA antibody (1:2000, Clone HA-7; Sigma-Aldrich), and monoclonal anti-Myc antibody (1:2000, Clone 9E10; Sigma-Aldrich).