Parp antibody

PARP antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), goat anti-mouse and goat anti-rabbit HRP-conjugated IgG from Bio-Rad (Hercules, CA), anti-nuclear factor erythroid related factor 2 (NRF2) antibody from Abcam (Cambridge, MA) and ECL Plus solution from Thermo-Fisher Scientific (Waltham MA, USA). CB-839 was supplied by Calithera, Inc. (South San Francisco, CA) and was dissolved either in DMSO for in vitro experiments (10 mM stock, stored at -20 ⁰C) or in vehicle for in vivo experiments. The in vivo vehicle consisted of 25% (w/v) hydroxypropyl-β-cyclodextrin in 10 mmol/L citrate (pH 2). The GLS inhibitor BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide) and all other reagents were purchased from Sigma (St. Louis, MO)

S-nitrosated proteome from cell extract was purified using a modified “biotin-switch” assay (13 (link),17 (link),18 (link)). After corresponding treatments on HEKn cells, the following steps were performed in the dark. Briefly, cells were harvested in RIPA cell lysis buffer (Thermo Scientific), sonicated, and centrifuged at 13,500 rpm for 15 min at 4 °C to remove cellular debris. 10 mM N-ethylmaleimide (NEM) was added and incubated for 1 h at room temperature to block all free Cys residues. Excess NEM in samples was removed by ice-cold acetone precipitation repeated three times for 20 min each. 1 mM ascorbic acid (Vc, Sigma-Aldrich) and 500 μM biotin-NEM (Thermo Scientific Pierce) were then added and incubated at room temperature for 1 h in order to reduce and label S-nitrosated Cys residues. Labeled S-nitrosated proteome was purified using streptavidin agarose beads (Thermo Scientific Pierce). Then the samples were resolved by electrophoresis through 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and probed with PARP antibody (Cell Signaling Inc. #9532). The membranes were developed using the SuperSignal chemiluminescent detection system (Thermo Scientific Pierce). Quantification of immunoblotting results was performed using software ImageJ. Statistical summaries were obtained from 3 independent samples.

Co-immunoprecipitation and western blot analysis was performed as described previously (Huang et al., 2015 (link); Power et al., 2015 (link)). Briefly, HeLa cells stably expressing Tat-Flag were treated with levosimendan (10 μM) for 24 hr. Total cell lysates were subjected to western blot analysis using Flag antibody (Cat. #14793, Cell signaling), PARP antibody (Cat. #9542, Cell signaling), or GAPDH antibody (Cat. #sc-47724, Santa Cruz). For Co-IP experiments, total cell lysates were immunoprecipitated using Protein A/G Magnetic Beads (Thermo scientific) with Flag antibody (Cat. #8146, Cell signaling). Immunoprecipitated proteins were subjected to western blot analysis using cyclin T1 antibody (Cat. #sc-10750, Santa Cruz).