Phenylmethanesulfonyl fluoride pmsf

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Gibco (United States). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (United States). RIPA lysis buffer and phenylmethanesulfonyl fluoride (PMSF) were obtained from Solarbio (China). The primary antibodies to cytochrome C, Bax, Bcl-2, caspase-3, caspase-9, HSP70, β-Tubulin, and β-actin were purchased from Cell Signaling Technology (United States). The secondary antibodies to HRP AffinPure goat anti-rabbit IgG and HRP AffinPure goat anti-mouse IgG were purchased from Proteintech (United States). The Alexa Fluor®488 goat anti-mouse/rabbit IgG and Alexa Fluor®568 goat anti-mouse/rabbit IgG were purchased from Invitrogen (United States). Triton X-100 and 4,6-dimethyl-2-phenylindole (DAPI) were purchased from Abcam (United Kingdom). Human hepatocellular carcinomas (HepG2) were obtained from American type culture collection (ATCC). The 2,2’-bis(anthracene-9,10-diylbis (methylene))-dimalonic acid (ABDA) was obtained from Shanghai Civi Chemical Technology Co.

(−)-epigallocatechin-3-O-gallate (EGCG) was purchased from Energy Chemical (Shanghai, China). 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-1,8-octanediol (12F) were purchased from Macklin Biochemical Co. Ltd. (Shanghai, China). RIPA lysis buffers and Phenylmethanesulfonyl fluoride (PMSF) were obtained from Solarbio (China). The primary antibodies to Bax, Bcl-2, PD-L1, STAT-1, P-STAT-1, IRF and β-actin were purchased from Cell Signaling Technology (United States). The secondary antibodies to HRP AffinPure goat anti-rabbit IgG and HRP AffinPure goat anti-mouse IgG were purchased from Proteintech (United States). The Alexa Fluor®488 goat anti-mouse/rabbit IgG and Alexa Fluor®568 goat anti-mouse/rabbit IgG were purchased from Invitrogen (United States). PBS, DMEM, and EDTA were purchased from WISENT. Triton X-100 and 4, 6-dimethyl-2-phenylindole (DAPI) were purchased from Abcam (United Kingdom). Human hepatocellular carcinomas (Hep3B) were obtained from American type culture collection (ATCC). All other reagents were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China) unless otherwise stated. Experimental data were obtained from Beiyu Biotechnology Co., Ltd. (Shanghai, China).

Cells (1 × 10^{7 (link)}) over-expressing Flag-GFP-14-3-3ε were treated with normal medium or medium containing Tp (20 nM). Twenty-four hours later, the cells were lysed with standard IP lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitor cocktail (Selleck Chemicals) and Phenylmethanesulfonyl fluoride (PMSF) (Solarbio Inc.,Beijing, China). Protein concentration was determined using BCA assays (Solarbio Inc.,Beijing, China). The cleared lysates were transferred to anti-Flag antibody beads or Protein A+G beads conjugated to IgG antibody (Cell Signaling Technology, #5750, Boston, USA) and incubated at 4°C overnight then washed four times with lysis buffer. For SDS-PAGE and Western blotting, the beads were incubated 3 min with SDS-PAGE loading buffer and boiled for 5 min. For liquid chromatography – tandem mass spectrometry (LC–MS/MS) analysis, the beads were eluted with 0.1 M Glycine (pH 2.6) and neutralized with 1 M Tris (pH 8.5) and collected. One sample of each group was used for LC – MS/MS and replicated for three times. Three samples of each group were used for western blotting verification and replicated for three times. A schematic representation is given in Figure 1.