The cells were lysed with RIPA buffer containing 1% protease inhibitor (Sigma‒Aldrich, St. Louis, MO, USA) on ice for 20 minutes, and the total protein in the lysate was separated by approximately 8% to 12% SDS–polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% skim milk at room temperature for 2 hours, followed by incubation with primary antibodies at 4°C overnight. The membranes were then incubated with 3% skim milk containing secondary antibodies at room temperature for 1 hour. Finally, the proteins were visualized using an ECL system (KeyGEN, Nanjing, China), and the protein density was analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used were as follows: Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA), phospho-cdc2 (Cell Signaling Technology, Danvers, MA, USA), cleaved PARP (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-9 (Cell Signaling Technology, Danvers, MA, USA), PI3K (Cell Signaling Technology, Danvers, MA, USA), phospho-PI3K (Cell Signaling Technology, Danvers, MA, USA), AKT (Abcam, Cambridge, UK), phospho-AKT (Abcam, Cambridge, UK), NF-κB (Servicebio, Wuhan, Hubei), phospho-NF-κB (Servicebio, Wuhan, Hubei), and actin (Proteintech, Beijing, China).
Ecl system
Manufactured by Keygen Biotech
Sourced in China
The ECL system is a laboratory equipment used for western blot analysis. It employs a chemiluminescent detection method to visualize and quantify protein targets.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Phospho cdc2, by Cell Signaling Technology (1 mentions)
Phospho akt, by Abcam (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Phospho pi3k, by Cell Signaling Technology (1 mentions)
Nf κb, by Wuhan Servicebio Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Pvdf membrane, by Keygen Biotech (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab214185, by Abcam (1 mentions)
Ab71495, by Abcam (1 mentions)
Ab133514, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
4 protocols using ecl system
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of Synovial Tissue and THP-1 Cells
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The synovium tissue and THP-1 cells were collected and lysed in a RIPA buffer (Beyotime, China). The total protein concentration was measured using a BCA protein assay kit (Beyotime, China). Samples containing ~50 mg protein was separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by the transference to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, MA, USA). Subsequently, PVDF membranes were blocked with 5% (w/v) non-fat milk in TBST buffer for 2 h at room temperature and treated with corresponding primary antibodies (1:500 to 1:1000) overnight at 4˚C. The membranes were washed three times with Tris buffer saline-Tween20 (TBST), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000 to 1:2000) for 2 h. Finally, protein bands were visualized with an enhanced chemiluminescence (ECL) system (Keygen Biotech, China) and scanned with a Chemiluminescence imaging system (Gel Catcher 2850, China). The relative optical densities of bands were analyzed with a ChemiScope analysis program.
3
Western Blot Analysis of Inflammasome Proteins
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Cell proteins were extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) with a protease inhibitor cocktail (Beyotime Institute of Biotechnology). First, equal amounts of proteins (50 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and then transferred onto polyvinylidene fluoride membranes. Next, 5% fat-free milk was used to block the membranes for 2 h, which were then incubated with primary antibodies: IL-1β (#12242, Cell Signalling Technology, CST), cleaved IL-1β (#52718, CST), IL-18 (ab71495, Abcam) 1:1000; caspase-1 (#24232, CST), cleaved caspase-1 (#89332, CST), NLRP3 (ab214185, Abcam), NEK7 (ab133514, Abcam) 1:500; ASC (#67824, CST) 1:200; and GAPDH (#5174, CST) 1:2000, overnight at 4 °C. Then the blots were incubated with secondary antibodies for 2 h. Finally, protein bands were visualized with an enhanced chemiluminescence (ECL) system (KeyGEN, Nanjing, China) and scanned with a chemiluminescence imaging system (Gel Catcher 2850, China). The grey values of bands were analysed with NIH ImageJ v1.52 software61 (link).
4
Western Blot Analysis of THP-1 Cells
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The THP-1 cells collected from each group were lysed in a RIPA buffer (Sigma, St. Louis, MO, USA). Samples containing approximately 50 mg protein was separated by 8–12% SDS-PAGE followed by the transference to polyvinylidene fluoride membranes (Millipore Corporation, MA, USA). Subsequently, PVDF membranes were treated with primary antibodies overnight at 4 °C after being blocked. The membranes were washed three times with Tris buffer saline-Tween20 (TBST), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 h. Finally, protein bands were visualized with an enhanced chemiluminescence (ECL) system (Keygen Biotech, China) and scanned with a Chemiluminescence gel imaging system (Tanon-5200Multi, China).
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