Ecl chemiluminescent detection system

Cells were collected and lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher, Rockford, IL, USA) containing 0.02% complete Protease Inhibitor EASY packs EDTA-Free (Roche Applied Science). Then the protein was separated using 12% polyacrylamide gel, and transferred to polyvinyl difluoride membranes. After blocking with 5% bovine serum albumin (BSA) in TBS/Tween20 (TBST) and incubated with specific primary antibodies at 4 °C overnight, followed by a 5 min wash with TBS-T, which was repeated three times. After this, the membranes were incubated with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h, followed a 5 min wash with TBST, which was repeated three times. Then the membranes were analyzed with the ECL chemiluminescent detection system (Bio-Rad).
The following antibodies were used in this study: anti-actin monoclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA) , anti-HIF1α polyclonal (Cell Signaling Technology), anti-VEGFA polyclonal, anti-cyclinD1 polyclonal (ProteinTech Group, Chicago, IL, USA) and anti-VHL polyclonal (Cell Signaling Technology).

All cells were seeded into six-well plates 24 h before protein extraction. Then, RIPA buffer containing proteinase inhibitors was used to lyse the cells. Western blot membranes were blocked in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk for 1 h. After blocking, primary antibodies against RASAL2 (CST, #82481, 1:1000), PI3K (CST, #4249, 1:1000), AKT (CST, #5741, 1:1000), p-AKT (S473, D9E, 1:1000) or cyclin D1 (WL01435a, 1:1000) were incubated with the membranes overnight at 4 °C. After the membranes were washed three times with TBST, they were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and visualised using an ECL chemiluminescent detection system (Bio-Rad Laboratories, Inc.). A monoclonal anti-β-actin antibody (CST, #3700) was used to normalise loading differences. Experiments were repeated three times.

Cells were lysed in RIPA buffer (50 mM Tris, pH 8.0,150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate) supplemented with protease inhibitor (1% inhibitors cocktail and 1 mM PMSF) (Roche Applied Science, Germany). Lysates were centrifuged at 12000 g for 15 min at 4°C, subjected to 8% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with the indicated primary antibody at 4°C overnight and were developed using the ECL chemiluminescent detection system (BioRad, USA). The experiment was repeated three times.

Cell lysates were separated using 10% SDS-PAGE and transferred onto Immobilon PVF-membranes (Millipore, USA) by electrotransfer. A protein size ladder (Thermo Fisher Scientific, USA) was used for size comparison. The primary antibodies used were as follows: cleaved caspase-3 (Cell Signaling Technology, USA), GSDME (Abcam, UK), caspase-1, GSDMD, IL-1β, IL-18 (all Abcam) and glyceraldehyde-3-phosphate dehydrogenase (ProteinTech Group, USA). Secondary horseradish peroxidase-coupled antibodies (KPL, USA) and Super ECL Plus chemiluminescent detection substrate (US Everbright and Immobilon Western) were used for detection. The protein bands were analyzed using an ECL chemiluminescent detection system (Bio-Rad, CA) and band intensity was quantified using ImageJ software version 1.8.0 (https://imagej.nih.gov/ij/).

Cell lysates were separated using 10% SDS-PAGE and transferred onto Immobilon PVF-membranes (Millipore, USA) by electrotransfer. A protein size ladder (Thermo Fisher Scientific, USA) was used for size comparison. The primary antibodies used were as follows: cleaved caspase-3 (Cell Signaling Technology, USA), GSDME (Abcam, UK), caspase-1, GSDMD, IL-1β, IL-18 (all Abcam) and glyceraldehyde-3-phosphate dehydrogenase (ProteinTech Group, USA). Secondary horseradish peroxidase-coupled antibodies (KPL, USA) and Super ECL Plus chemiluminescent detection substrate (US Everbright and Immobilon Western) were used for detection. The protein bands were analyzed using an ECL chemiluminescent detection system (Bio-Rad, CA) and band intensity was quantified using ImageJ software version 1.8.0 (https://imagej.nih.gov/ij/).

The cell lysates were prepared using RIPA buffer (50 mM Tris, PH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate) containing proteinase inhibitors (1% inhibitors cocktail and 1 mM PMSF) (Roche Applied Science, Germany) for 10 min in ice and centrifuged at 12000g for 15 min at 4°C. The samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin, the membranes were incubated with primary antibody at 4°C overnight. Next, the membranes were washed with TBST buffer for 3 times and incubated with peroxidase-conjugated secondary antibody for 1h at room temperature. After washing with TBST buffer for 3 times again, the membranes were visualized by using an ECL chemiluminescent detection system (Bio-rad, USA).

As previously described with modification [1 (link)], the cells and tissues were collected and lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher, Rockford, IL, USA), containing 1% phenylmethylsulfonyl fluoride (PMSF). Protein concentration was quantified with the BCA Protein Kit (Beyotime, China). Lysates (10 μg proteins) were separated by 10% SDS-PAGE gels and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% Bovine Serum Albumin in TBS/Tween20 (TBST) for 2 hours at room temperature, and then probed with primary antibodies against proteins of interest overnight at 4°C. After incubated with secondary antibodies for 1 hour at room temperature, protein signal was detected with the ECL chemiluminescent detection system (Bio-Rad), and protein levels were normalized to β-actin.