Protein a or g magnetic beads

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.

Protein lysates were prepared using RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors. The whole-cell lysates were incubated with 100 μL of protein A or G magnetic beads (Santa Cruz Biotechnology, USA) prebound with anti-EIF1AX (Thermo Fisher Scientific, Cat#HPA002561), anti-FLAG (Millipore, Cat#F7425), anti-IPO13 (Novus Biologicals, Cat#NBP1-31508), anti-XPO1 (Santa Cruz Biotechnology, Cat#sc-74454), normal rabbit IgG control, or normal mouse IgG control. The immunoprecipitates were analyzed using western blotting after elution in sample buffer at 95°C.