Rabbit polyclonal anti bcl 2

INS-1 cells and mouse pancreases were lysed in radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology Corporation, Rockford, IL, USA), and quantified for total protein using a Micro BCA Protein Assay Kit (Pierce Technology). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was then blocked with 5% skimmed milk. The membrane was incubated overnight at 4 °C with one of the following primary antibodies: mouse monoclonal anti-TRAIL, goat polyclonal anti-DR5, goat polyclonal anti-DcR1, mouse monoclonal anti-Bax, rabbit polyclonal anti-Bcl-2, mouse monoclonal anti-NF-κB, goat polyclonal anti-α-tubulin, mouse monoclonal anti-β-actin (all from Santa Cruz Biotechnology, Dallas, TX, USA), or mouse monoclonal anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA). The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). Protein bands were detected by enhanced chemiluminescence (Pierce Biotechnology). The band intensities of the proteins were analyzed using ImageJ densitometry software (version 1.43; National Institute of Health, Bethesda, MD, USA).

Bafilomycin A1, Rapamycin, and 8-Bromoadenosine-3',5'-cyclic monophosphate sodium salt (8-Br-cAMP) were purchased from EMD Millipore (Darmstadt, Germany); 3-isobutyl-1-methylxanthine (IMBX) was purchased from Cayman Chemical (Ann Arbor, MI). Fluorescent probes Mitotracker Green FM, Lysotracker Red DND-99, Dihydroethidium (DHE), and MitoSOX Red mitochondrial superoxide indicator were purchased from Molecular Probes (Grand Island, NY). JC1 and DCF were purchased from Sigma (St Louis, MO), and Annexin V was purchased from BD Biosciences (San Jose, CA). Antibodies were obtained as follows: mouse monoclonal anti-BNIP3 (Sigma, St Louis, MO, B7931), mouse monoclonal anti-His-tag (Qiagen, Valencia, CA, 34650), rabbit polyclonal anti-VDAC1 (Novus Biologicals, Littleton, CO, NB 100–695), rabbit monoclonal anti-PKA substrate (Cell Signaling Technology, Danvers, MA, 9624S), rabbit polyclonal anti-LC3 (Novus Biologicals, Littleton, CO, NB 100–2220), mouse monoclonal anti-SQSTM1 (Abcam, Cambridge, MA, ab56416), mouse monoclonal anti-OPA1 (BD Biosciences, 612606), rabbit polyclonal anti-BCL2 (Santa Cruz Biotechnology, Inc., Dallas, TX, sc-492), mouse monoclonal anti-MT-CO2 (Abcam, ab3298), rabbit monoclonal anti-MT-CO2 (Abcam, ab79393), rabbit polyclonal anti-ACTB (actin, beta) (Cell Signaling Technology, Danvers, MA, 4968), and mouse monoclonal anti-GAPDH (Acris, San Diego, CA, ACR001PT).