Total protein samples were extracted from cells after incubating with valsartan, tetrandrine and Y-27632 for 48 h or with U73122 for 10 min. RIPA lysis buffer (Beyotime) with 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime) was used for cell lysis. The supernatant was transferred to a centrifuge tube for vortexing. Protein lysates were centrifuged at 4°C, 12,000 × g for 10 min. Protein samples were subjected to 10% SDS-PAGE for separation and then transferred onto polyvinylidene difluoride (PVDF) membranes according to standards methods. PVDF membranes were blocked with skim milk powder, and incubated with primary antibodies. against TRPC6 (1: 1000, Santa Cruz), Synaptopodin (1: 100; Santa Cruz), Nephrin (1: 100; Proteintech), ROCK1 (1: 1000; Proteintech), anti-RhoA antibody (1: 1000; Proteintech, Wuhan, China), and β-actin (1: 1000; Santa Cruz) at 4°C overnight. Activated RhoA was determined by measuring membrane-bound RhoA (GTP-RhoA) using the GST rhotekin-Rho binding domain that specifically pulls down activated RhoA, Secondary antibodies were peroxidase AffiniPure donkey anti-mouse or goat anti-rabbit IgG (H+L; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The blots were developed using ECL system (Share-bio, Shanghai, China) and analyzed using the TanonImage software (Tanon, Shanghai, China).
Ecl system
Manufactured by Share-Bio
Sourced in China
The ECL (Enhanced Chemiluminescence) system is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate that reacts with the enzyme-labeled secondary antibody, emitting light in proportion to the amount of target protein present. This system provides a sensitive and reliable method for visualizing and analyzing protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Immobilon pvdf membrane, by Merck Group (3 mentions)
Synaptopodin, by Santa Cruz Biotechnology (2 mentions)
Rock1, by Proteintech (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Trpc6, by Santa Cruz Biotechnology (1 mentions)
Anti rhoa antibody, by Proteintech (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Donkey anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Anti trpc6, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Abcam (1 mentions)
Protease inhibitor, by Selleck Chemicals (1 mentions)
Quickblock, by Beyotime (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
5 protocols using ecl system
1
Analysis of RhoA/ROCK Signaling Pathway
2
Immunoblotting Protein Extraction Protocol
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Proteins were extracted from both cell lines and tissues using RIPA lysis buffer (Thermo Fisher Science, USA) in cold conditions. After centrifugation at 15000g for 10 min, the protein samples were subjected to immunoblotting. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Science, USA). Subsequently, the protein samples were loaded onto PAGE gels (Epizyme Biomedical Technology, Shanghai, China) and transferred to 0.22 µm Immobilon PVDF membranes (Millipore Sigma, USA). After blocking with 5% milk, the membranes were incubated in primary antibodies at appropriate dilutions overnight at 4 °C. Next, secondary antibodies (anti-Rabbit IgG) were applied at room temperature for approximately 1 h, and the immunoreactivity was visualized using an ECL system (Share-bio, Shanghai, China). The dilution factor for primary antibodies against pan-kla (#PTM-1401RM, PTMBio, Hangzhou, China) was 1:1000, and for Histone H3 (#P30266, Abmart, Shanghai, China) was 1:2000. All secondary antibodies were used at a dilution of 1:5000.
3
Western Blot Analysis of Cellular Proteins
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Proteins were extracted from cell lines using RIPA buffer (Thermo) containing a protease inhibitor. Protein samples were loaded onto PAGE gels (Epizyme Biomedical Technology, Shanghai, China) and transferred to 0.2 µm immobilon PVDF membranes (Millipore Sigma). Subsequently, membranes were incubated with primary antibodies (anti-CD63, anti-TSG101, anti-NAT10, and the loading control anti-β-actin) at appropriate dilutions at 4 °C overnight. After washing, the membranes underwent incubation with secondary antibodies at a 1:5000 dilution at room temperature for 1 h. Finally, blots were visualized using an ECL system (Share-bio, Shanghai, China).
4
Western Blot Analysis of Kidney Protein Markers
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Total protein was extracted from MPC5 podocytes and kidney tissues of rats. For sufficient cell lysis, RIPA lysis buffer (Beyotime, China) was added, and then, the supernatant was extracted after centrifugation at 12,000 rpm for 10 min. Protein samples were separated with 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with skim milk powder and then incubated with primary antibodies to anti-TRPC6 (1 : 1,000; Santa Cruz, USA), RhoA (1 : 1,000; Proteintech, Wuhan, China), ROCK1 (1 : 3,000; Proteintech), synaptopodin (1 : 1,000; Santa Cruz), and GAPDH (1 : 10,000; Abcam, UK). Membranes were then incubated with donkey anti-mouse IgG (H+L; Jackson ImmunoResearch Laboratories, USA) secondary antibody. ECL system (Sharebio, China) was used for band detection and protein bands were analyzed by the Tanon Image software (Tanon, China).
5
Immunoblotting Protein Expression Analysis
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The proteins were acquired from cell lines using RIPA buffer (Thermo) containing a protease inhibitor (Bimake), and then immunoblotting was performed. A bicinchoninic acid protein assay kit was applied to quantify the protein content of the cell lysates. Protein samples were loaded into PAGE gels (Epizyme Biomedical Technology, Shanghai, China) and transferred to 0.2 µm immobilon PVDF membranes (Millipore Sigma). After blocking with quick blocking buffer (QuickBlock™, Beyotime ), membranes were covered with primary antibodies in proper dilutions overnight at 4 °C and secondary antibodies were incubated at room temperature for approximately 1 hour and then visualized by an ECL system (Share-bio, Shanghai, China). The dilution factor of primary antibodies against BST2 (ab88523) and PD-L1(ab213480) was 1:1000, and GAPDH (Proteintech, 60004-1-Ig) was 1:5000. Secondary antibodies included anti-mouse IgG (Proteintech, SA00001-1) and anti-rabbit IgG (Proteintech, SA00001-2 ), and all the secondary antibodies were diluted at 1:5000.
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