Mouse anti par antibody

The following reagents were obtained commercially: rabbit anti-caspase-3 from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-HMGB1 from Abcam (Cambridge, UK); mouse anti-β-actin, H₂O₂, t-BHP, propidium iodide (PI), STS, Necrostatin-1 (Nec-1), 3-Aminobenzamide (3AB), 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), poly-l-lysine, nicotinamide adenine dinucleotide (NAD), SI and 50% glutaraldehyde from Sigma-Aldrich (Saint Louis, MO, USA); horseradish peroxidase (HRP)-conjugated anti-mouse antibody, HRP-conjugated anti-rabbit antibody, fluorescein (FITC)-conjugated anti-mouse antibody and 4′,6-diamidino-2-phenylindole (DAPI) from Thermo Fisher Scientific (Rockford, IL, USA); mouse anti-RIPK1 antibody, mouse anti-PARP-1 antibody, and Annexin V from BD Biosciences (San Jose, CA, USA); rabbit anti-RIPK3 antibody and mouse anti-AIF antibody from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-PAR antibody from Trevigen (Gaithersburg, MD, USA); mouse anti-PAR antibody from Enzo Life Sciences (Farmingdale, NY, USA); z-VAD-fmk from Millipore (Darmstadt, Germany); olaparib from Selleck Chemicals (Houston, TX, USA); UPF-1069 and XAV-939 from Tocris Bioscience (Minneapolis, MN, USA); and MNNG from Tokyo Chemical Industry (Tokyo, Japan).

Cells were fixed with 4% paraformaldehyde (PFA) dissolved in deionized water for 15 min and permeabilized in PBS containing 0.1% Triton X-100. Following rinsing twice with phosphate buffer saline (PBS) containing 0.5% Tween 20 (PBST), cells were blocked in PBS containing 5% goat serum for 30 min. Cells were incubated with the primary rabbit anti-TRPM2 antibody (Bethyl) at a dilution of 1:1500 or mouse anti-PAR antibody (Enzo;1: 500) overnight at room temperature and, after extensive washing in PSBT, incubated with the secondary FITC-conjugated goat anti-rabbit IgG antibody (Sigma; 1:1000) or anti-mouse IgG antibody (Sigma; 1: 1000) for 1 hr at room temperature. After washing with PBS and rinsing in water, coverslips were mounted using florescent mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). All images were captures using an Olympus IX51 microscope, a digital camera and Cell^F software (Olympus). The intensity of the fluorescent was quantified using ImageJ and at least 75 cells were examined in each well.