Ha tag mouse mab

The apoptosis inducer ATP was purchased from Sigma-Aldrich (St. Louis, USA), the P2X1 antagonist NF449 from Tocris Bioscience (Minneapolis, USA), Fluo-4 AM from Dojindo Laboratories (Mashiki-machi, Japan), cell mitochondria isolation kit from Beyotime Biotechnology (Beijing, China) and Lipofectamine® 3000 transfection kit from Thermo Fisher Scientific (Waltham, USA). An isothiocyanate (FITC)-Annexin V/propidium iodide (PI) apoptosis detection kit, TUNEL in situ cell death detection kit and JC-1 mitochondria membrane potential detection kit were purchased from KeyGen (Nanjing, China); protein A-agarose immunoprecipitation reagent and normal rabbit IgG from Santa Cruz Biotechnology (California, USA); HA-tag rabbit mAb, HA-tag mouse mAb, β-actin rabbit mAb, caspase-3 rabbit mAb, caspase-7 rabbit mAb, caspase-9 rabbit mAb and PARP rabbit mAb from Cell Signaling Technology (Boston, USA); Bax rabbit mAb, Bcl2 rabbit mAb, Cyt C rabbit mAb, P2X1 rabbit pAb and cytochrome C oxidase IV (COXIV) rabbit mAb from Abcam (Cambridge, UK); and DDDDK-Tag Mouse mAb from Abclonal (Boston, USA).

Briefly, cells were seeded in a 96-well plate to reach 50%–60% confluency overnight. Cells were washed and fixed with 100 μL 4% paraformaldehyde in PBS for 10 min at 37°C, permeabilized with 100 μL 1% Triton X-100 in 1× PBS for 15 min at 37°C, and blocked with 1% BSA in 1× PBS for 1 h at 37°C. Cells were then incubated with primary antibody (HA-Tag Mouse mAb, 1:100 dilution; Cell Signaling), washed and incubated with secondary antibody (Cy3 conjugated goat anti-mouse, 1:2,000 dilution; Jackson ImmunoResearch). Samples were washed and images were obtained from an EVOS fluorescence microscope (ThermoFisher Scientific).