Ha tag rabbit mab

Protein samples mixed with 5 × loading buffer were boiled at 100℃ for 5 minutes. Then cooled down the samples and subjected them into sodium dodecyl sulfate polyacrylamide gel, electrophoresed and transferred the sample to PVDF membrane. Then blocked the membrane with 5% skimmed milk for 1 hour, and then incubated the membrane with primary antibodies, at 4℃ overnight. Washed the membrane, incubated it into secondary antibodies, and washed it again for 3 times. At last, detected the signals with ChemiDoc (Bio-Rad, CA, USA). The primary antibodies used in this study were as follow: rabbit anti-FLOT1(Proteintech, 15571-1-AP, 1:1000), rabbit anti-ERK1/2 (Proteintech, 51068-1-AP, 1:1000), rabbit anti-p-ERK1/2 (Thr202/Tyr204) (Proteintech, 28733-1-AP, 1:1000), rabbit anti-p130 Cas (Cell Signaling Technology, #13846, 1:1000), rabbit anti-p-p130 Cas (Tyr410) (Cell Signaling Technology, #4011, 1:800), HA-Tag Rabbit mAb (Cell Signaling Technology, #3724, 1:1000), DYKDDDDK Tag Rabbit mAb(Cell Signaling Technology, #14793, 1:1000) antibodies.

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).