Protease inhibitor cocktail

RIPA buffer (Thermo Scientific, 89900) was used to lyse cells for the purpose of isolating overall protein and supplemented with protease inhibitor cocktail (Bestbio, Shanghai, China) and Phenylmethanesulfonyl fluoride (PMSF) (Bestbio, Shanghai, China). The separation of samples was carried out using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane, which was stalled using 5% skim milk at 4°C overnight and incubated overnight with specific antibodies to METTL14 (1:1,000, ab220030, Abcam), PTEN (1:1,000, #9188, CST); AKT (1:1,000, #4685, CST); Phospho-Akt (Ser473, 1:1,000, #4060, CST). Following, secondary antibodies at 37°C for 1 h were combined with membranes, and imbibed in electro-chemiluminescence solution for imaging. An evaluation of relative protein level was performed after all the earlier mentioned steps had been successfully completed.

Cellular proteins were extracted by using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (BestBio, Shanghai, China). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer (5% nonfat milk powder in TBS containing 0.1% Tween) for 2 h. Then, the membranes were incubated with various primary antibodies followed by the appropriate horseradish peroxidase- (HRP-) conjugated secondary antibodies. Additionally, enhanced chemiluminescence (ECL) (Millipore, Billerica, MA, USA) was used to identify immunoreactive bands. All experiments were repeated three times. The antibodies used in this study are listed in Tables 1 and 2.

To isolate overall protein, CRC cells received lysing process with radioimmunoprecipitation assay (RIPA) buffer (89900; Thermo Scientific) containing phenylmethanesulfonyl fluoride (PMSF; Bestbio) and protease inhibitor cocktail (Bestbio, Shanghai, China). The protein samples were extracted using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and placed on nitrocellulose membranes, blocked with 5% skim milk at 4°C throughout the night, and subsequently cultured throughout the night with antibodies specific for YAP1 (1:1,000, ab52771, Abcam, Cambridge, MA, USA), CTGF (1:1,000, ab231824, Abcam); AREG (1:1,000, ab180722, Abcam); E-carherin (1:2,000, ab15148, Abcam); vimentin (1:2,000, ab8069, Abcam); Snail (1:1,000, ab229701, Abcam); Slug (1:1,000, ab51772, Abcam), and Twist (1:500, ab175430, Abcam). The membranes were subsequently cultivated with secondary goat anti-rabbit IgG antibody under conjugation with horseradish peroxidase (1:100, ab109489, Abcam) at 37°C for 1 h and then immersed in electro-chemiluminescence solution for imaging after which the relative protein levels were analyzed as previously described (17 (link)).

The downstream of PD-1 signaling included attenuating the activation of phosphatidylinositol 3-kinase (PI3K)/AKT pathway which can improve T lymphocyte metabolism, regulate cell cycle and apoptosis, and affect T lymphocyte activation and immune function (25 (link)–27 (link)), the protein expression of PI3K and AKT was analyzed. CD8⁺ T cells cultured with 100 nM decitabine or PBS were lysed in ice-cold RIPA buffer containing a protease inhibitor cocktail (BestBio, Shanghai, China) and concentrations of the protein were determined by BCA assay kit (BestBio). After denatured at 95°C for 5 min, the protein samples were loaded and separated by SDS-PAGE and transferred onto a NC membrane. Non-specific binding sites were blocked with 5% (w/v) fetal bovine serum at room temperature for 2 h. The membrane was incubated with polyclonal rabbit anti-human β-Actin (Cell Signaling Technology, NJ, USA), polyclonal rabbit anti-human PI3K, p-PI3K, AKT, and p-AKT antibodies (Cell Signaling Technology, NJ, USA). The blots were further incubated with HRP-conjugated secondary antibodies and washed three times in PBST for 5 min each time. Protein expression was detected by ECL and photographed by Bio-Spectrum Gel Imaging System (UVP, USA).

AGS and BGC-823 cells were cultured in RPMI 1640 medium at the mid-log phase and then incubated with pholidonone at 0, 5, 10, 20, or 40 μM for 48 h. After the treatments, cells were washed by pre-cooled PBS and lysed in RIPA buffer supplemented with 1mM PMSF and protease inhibitor cocktail (Bestbio) at a dilution of 1:100. Protein concentration was determined using the BCA^TM protein assay kit (Pierce). Protein samples (50 μg) were loaded on SDS-PAGE and then transferred to a PVDF membrane (Millipore). After blocked with 5% nonfat dry milk in TBST for 2 h, the membranes were incubated with primary antibodies (1:1000, CST, including anti-BiP, anti-Calnexin, anti-Ero1-Lα, anti-IRE1α, anti-CHOP, anti-PERK, anti-PDI, and anti-cleaved-caspase-3) overnight at 4°C. The blots were washed and incubated with HRP-linked secondary antibodies (1:2000, CST) for 2 h at room temperature. Membranes were again washed three times with TBST and visualized using enhanced chemiluminescence substrate (Immobilon^TM Western; Millipore). TM was used as a positive control.

After treatments, expressions of Rho GTPases in HUVECs were performed by Western Blot. Cells were washed and then lysed on ice for 10 min in RIPA Lysis Buffer (Beyotime, Jiangsu, China) with an addition of protease inhibitor cocktail (1:100, BestBio Science, Shanghai, China), phosphatase inhibitor cocktail (1:100; BestBio Science, Shanghai, China) and 10 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration was measured by a Protein Determination Kit (Cayman Chemical, Ann Arbor, MI, USA). Proteins were size fractionated using SDS-PAGE and electrotransferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA), and hybridized with monoclonal antibodies (Santa Cruze, USA) including mouse anti-RhoA antibody (1:500), Rabbit anti-Rac1 antibody (1:200) and mouse anti-Cdc42 antibody (1:200). Detection was carried out using peroxidase-conjugated secondary antibodies (goat anti-mouse or goat anti-rabbit, 1:5,000) and enhanced chemiluminescence reagents (BeyoECL Plus, Beyotime, Jiangsu, China). Blots were imaged by Molecular Image^® ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad, Hercules, CA, USA). Quantitative data were obtained by using ImageJ 1.50b Gel Analyzer.