Plko bim shrna

MIN6 cell culture, RNA isolation, and first-strand cDNA synthesis, mouse pancreatic islet isolation, and preparation of pLKO.1-Pdx1 short hairpin RNA (shRNA) lentivirus all were performed as previously described (10 (link)). Applied Biosystems (Foster City, CA) TaqMan assay numbers were as follows: Hmbs, Mm00660262_g1; Pdx1, Mm00435565_m1; Bim, Mm00437796_m1; and Puma, Mm00519268_m1. The pLKO-Bim shRNA (TRCN0000009692 and 9693), Puma shRNA (TRCN0000009712 and 9713), and Pdx1 (8 (link)) and lentiviral vectors (TRCN0000086031) were purchased from Thermo Scientific. Bim shRNA targets all Bim isoforms. Lentivirus was added to the medium on day 1. The blots were probed with antibodies against Pdx1 (07-696; Millipore), Puma (7467; Cell Signaling), Bim (202000; Calbiochem), cleaved caspase3 (9661; Cell Signaling), and actin (A-2066; Sigma). Antibody detection was accomplished using an enhanced chemiluminescence method and LAS-3000 imaging system (FUJIFILM).

MIN6 cell culture, RNA isolation and first-strand cDNA synthesis, and preparation of pLKO.1-Pdx1 short hairpin RNA (shRNA) lentivirus all were performed as previously described (16 (link)). TaqMan assay numbers (Invitrogen) were as follows: mouse actin B, 4352933; IRS2, Mm003038438_m1; Bim, Mm00437796_m1; and Puma, Mm00519268_m1. The pLKO-Bim shRNA (TRCN0000009692), IRS2 shRNA (TRCN00000055110), and FoxO1 (TRCN0000054880) lentiviral vectors were purchased from Thermo Scientific. Lentivirus was added to the medium on day 1. The blots were probed with antibodies against IRS2 (3089; Cell Signaling), Puma (7467; Cell Signaling), cleaved caspase-3 (9661; Cell Signaling), FoxO1 (2880; Cell Signaling), p-AKT and AKT (9916; Cell Signaling), Bcl-xL (2762; Cell Signaling), Bcl-2 (554218; Pharmingen), BAD (sc-943; Santa Cruz Biotechnology), Mcl-1 (sc-819; Santa Cruz Biotechnology), Bim (202000; Calbiochem), and β-actin (A-2066; Sigma-Aldrich).