Shshc gipz lentiviral shrna

Media DMEM and F12-B, minimal essential medium amino acids, penicillin/streptomycin, fetal bovine serum were purchased from GIBCO (Grand Island, NY, USA). Insulin and specific brown fat inhibitor SR59230A were from Sigma (St. Louis, MO, USA). Antibodies were from Cell Signaling Inc. (Danvers, MA, USA) except: rabbit anti-Shc and anti-Cyt-C antibody were from BD Biosciences (San Diego, CA, USA); rabbit antiphospho-PDH, PCX, UCP3, goat anti-GCKR, mouse anti-ACADVL were from Novus Biologicals (Littletown, CO, USA); anti-Tubulin – Sigma; rabbit anti-UCP1 and CPS1, mouse anti-PDH, MCAD, and ACAA1 were from Abcam Inc. (Cambridge, MA, USA); rabbit anti CPT2, PFKL, HK were from Epitomics Inc. (Burlingame, CA, USA); rabbit anti-GCK – from LifeSpan BioScience Inc. (Seattle, WA, USA); goat anti-PFKM were from GenWay Biotech Inc. (San Diego, CA, USA); goat anti-UCP2 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); the secondary infrared dye 700 and 800 labeled antibody were from Li-Cor Biosciences (Lincoln, NE). Trans-Lenti shRNA packaging plasmids, shShc-GIPZ lentiviral shRNA and Nonsilencing-GIPZ lentiviral shRNAmir control plasmids were from Thermo Scientific Inc. (Pittsburgh, PA, USA).