MCF-7 cells were transfected with si-NC or si-svAC3-33, pEGFP-C3 or pEGFP-C3-svAC3-33, and MCF-7 cells were incubated at 37°C for 48 h, then total cell extracts and the western blotting was performed as previously described (20 (link)). The total protein concentration was determined using the BCA protein assay kit (Sangon Biotech Co., Ltd.). Specific antibodies for the following proteins were used: Anti-green fluorescent protein (1:5,000; cat. no. D110008; BBI Solutions), anti-c-Fos (1:500; cat. no. D120415; BBI Solutions), anti-c-Jun (1:500; cat. no. D155181; BBI Solutions) and anti-β-actin (1:50,000; cat. no. AC026; ABclonal Biotech Co., Ltd.). Bands were visualized using an ECL kit (Sangon Biotech Co., Ltd.).
Ecl kit
Manufactured by Sangon
Sourced in China
The ECL kit is a laboratory equipment designed for protein detection and quantification. It utilizes enhanced chemiluminescence (ECL) technology to enable sensitive and accurate analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Sangon (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Sangon (2 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
β catenin, by Thermo Fisher Scientific (1 mentions)
Gapdh antibody, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Precast gel, by Beyotime (1 mentions)
Bca kit, by Sangon (1 mentions)
Skimmed milk, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Abcam (1 mentions)
Anti caspase 3, by Thermo Fisher Scientific (1 mentions)
Anti cd63, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Sangon (1 mentions)
12 protocols using ecl kit
1
Transcriptional Regulation of svAC3-33 in MCF-7 Cells
2
Western Blotting Analysis of Apoptosis and Proliferation Markers
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Transfected HEC1A and Ishikawa cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology). Total protein concentration was qualified with a BCA Protein Assay kit (Sangon Biotech Co., Ltd.). Next, protein samples (~1,000 ng/lane) were separated by 12% SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies [cleaved caspase-3, 1:500, cat. no. ab32042; Bcl-2, 1:1,000, cat. no. ab32124; proliferating cell nuclear antigen (PCNA), 1:1,000, cat. no. ab29; AKT3, 1:2,000, cat. no. ab152157; β-actin, 1:5,000, cat. no. ab6276] at 4°C overnight. Anti-rabbit (cat. no. ab150077, for cleaved caspase-3, Bcl-2, AKT3, β-actin) and anti-mouse IgG (cat. no. ab150113, 1:10,000, for PCNA) were used as the secondary antibody at room temperature for 2 h. All the antibodies was purchased from Abcam (Shanghai, China). ECL kit (Sangon Biotech Co., Ltd.) was used for chemiluminescent detection of immobilized proteins according to the manufacturer' protocol. All antibodies were obtained from Abcam. The protein bands were quantified with ImageJ V1.8.0 software (National Institutes of Health).
3
Cardiac Tissue Western Blotting Analysis
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Western blotting analyses of cardiac tissue and H9C2 cells were performed as described. Briefly, total proteins of heart tissues or H9C2 cells were extracted with RIPA lysis buffer (R0020, Solarbio, China) containing protease inhibitor (04693116001, Roche, Germany). Then protein concentration was quantified by the BCA protein assay kit (C503051, Sangon, China). 30 μg of total protein samples from different groups were first electrophoresed through 10% SDS-PAGE gels and then transferred onto the nitrocellulose membranes (F619511, Sangon, China). After blocked with 5% skim milk in TBST at room temperature for 1 h, membranes were washed and incubated with the following primary antibodies overnight (as shown in Table 2
4
Western Blot Analysis of Cell Signaling
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The cells were lysed by RIPA buffer (Beyotime, China). The total protein was quantified by BCA kit (Sangon, China). The total protein with amount of 20 μg was separated by 4–20% precast gel (Beyotime, China) and transferred to nitrocellulose membrane (Sigma, USA). The membrane was blocked by 5% BSA (Beyotime, China) at 37°C for 1 h, incubated with diluted primary antibody at 4°C overnight, incubated with secondary antibody at 37°C for 1 h, successively. At last, the protein bands were developed by ECL kit (Sangon, China) and quantified by Image J 1.8.0 (NIH, USA). The primary antibodies were listed as follows: PLK4 antibody (1:1000, Invitrogen, USA), GSK3β antibody (1:3000, Invitrogen, USA), p-GSK3β (1:1000, Invitrogen, USA), β-catenin (1:1000, Invitrogen, USA), GAPDH antibody (1:5000, Invitrogen, USA). The secondary antibody was HPR linked goat anti-rabbit IgG antibody (1:200000, Invitrogen, USA).
5
Protein Extraction and Western Blot
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The medium was removed and the adherent cells were washed twice with precooled PBS (4°C). A total of 100 μl modified radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF; 1:200) was added to a 60-mm culture dish and the cells were scraped on ice. The cell lysates were collected into EP tubes and reacted in an ice bath for 30 min, with shaking for 10 s every 5 min. The cell lysate was centrifugated (12,000 r.p.m., 4°C, 10 min) and the supernatant was taken. The protein concentration was measured with the BCA protein assay kit (Beyotime, Shanghai, China). The protein sample was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Sigma, MA, USA) by gel electrophoresis, and then the PVDF membrane was blocked with 5% skimmed milk (Beyotime, Shanghai, China) for 2 h. The membrane was incubated with primary antibody solution (Abclonal, Wuhan, China) at 4°C overnight. After washing three times with trisbuffered saline with 0.1% Tween ® 20 (TBST), the membrane was probed with goat antirabbit or mouse secondary antibodies (CST, MA, USA) at room temperature for 2 h, and then detected with an ECL kit (Sangon, Shanghai, China).
6
Western Blot Analysis of Viral Nucleocapsid
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NP expression in infected cells was analyzed by western blot. Protein samples were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, USA), before being blocked with 6% Rapid Block Buff II (Sangon Biotech, China) at room temperature for 10 min. The blot was probed with the antibody against the viral nucleocapsid protein (Cambridgebio, USA) and the horseradish peroxidase-conjugated Goat Anti-Rabbit IgG (Abcam, USA) as the primary and the secondary antibodies, respectively. Protein bands were detected by chemiluminescence using an ECL kit (Sangon Biotech, China).
7
Exosome Protein Analysis in Parkinson's Disease
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The protein levels were detected as the previously performed [35 (link)]. The exosome samples (exosome from control or PD groups, Exo-control or Exo-PD) were lysed in lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1 mM PMSF with protease inhibitor mixture, pH 8.0. The supplement was collected after being centrifugated at 14,000 g for 10 min at 4 °C. The loading buffer was added to the protein samples and boiled for 5 min at 98 °C. The protein samples were resolved by 12% SDS-PAGE and then transferred to PVDF membranes for immunoblotting. PVDF membranes were blocked in blocking buffer (1 X TBS containing 5% skim milk) at room temperature for 1 h, followed by diluting with primary antibodies and HRP-conjugated secondary antibodies. The immunoblotting bands were visualized with an ECL kit (Sangon). Images were captured using a chemiluminescence imaging analysis system (Tanon, 5200) and analyzed with the ImageJ.
Antibodies: Anti-ZNF865 (1:1000, PA5-49280, Thermo), Anti-Caspase 3 (1:1000, 700182, Thermo), Anti-CD63 (1:1000,10628D, Thermo), Anti-GAPDH (1:1000, MA1-16757, Thermo), HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (1:5000, Proteintech).
8
Western Blot Analysis of WAVE3 Protein
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Total protein was extracted from cells transfected with mimics and mimic negative controls using RIPA lysis buffer (Sangon Biotech Co., Ltd.) and quantified using a bicinchoninic acid protein assay kit (Sangon Biotech Co., Ltd.), according to the manufacturer's protocol. Subsequently, proteins (25 µg/lane) were separated via 12% SDS-PAGE and transferred onto PDVF membranes. After blocking with 5% non-fat dry milk for 2 h at room temperature, the membranes were incubated with the anti-WAVE3 primary antibody (1:1,000, cat. no. 2806S; Cell Signaling Technology, Danvers, Inc.) and anti-GAPDH primary antibody (1:6,000, cat. no. G8795; Sigma-Aldrich; Merck KGaA), overnight at 4°C. Subsequently, the membranes were washed with TBST and incubated with the HRP-conjugated goat anti-rabbit secondary antibody (1:1,000, A0208, Beyotime Institute of Biotechnology) for 2 h at room temperature. Protein band images were visualized with an ECL kit (Sangon Biotech Co., Ltd.) and captured using a chemiluminescence imaging system (EMD Millipore) and then analyzed with ImageJ (version 1.8.0; National Institutes of Health). GAPDH was used as the loading control.
9
Protein Expression Analysis in Muscle Cells
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After 48-h treatment, the total protein of gastrocnemius samples and C2C12 myotubes were isolated by RIPA (Beyotime Biotechnology, Haimen, China). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted, and the protein was transferred to polyvinylidene fluoride membranes (BioSharp, Tallinn, Estonia). After being blocked, the membranes were incubated with primary and secondary antibody, successively. Finally, an ECL Kit (Sangon Biotech, Shanghai, China) was used for the detection of protein bands. The antibodies were purchased from Abcam (Cambridge, UK), including p-adenosine 5’-monophosphate-activated protein kinase (AMPK; 1:1,000 and 1:2,000), silent information regulator factor 2-related enzyme 1 (SIRT1) (1:2,000), hypoxia-inducible factor-1α (HIF-1α; 1:500), p-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K; 1:2,000 and 1:1,000), p-protein kinase B (AKT; 1:2,000 and 1:1,000), cleaved caspase-3 (C-caspase 3) (1:1,000), B-cell lymphoma-2 (Bcl2, 1:1,000), GAPDH (1:5,000), and goat anti-rabbit immunoglobin G (IgG; H + L) horseradish peroxidase antibody (1:10,000).
10
Protein Expression Analysis of Stromal Cells
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As described previously (Lin et al. 2018) . Proteins were extracted from cultured stromal cells with lysis buffer. The BCA reagent kit (Applygen, Beijing, China) was used to measure the concentration of proteins. The protein samples were separated by 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore). PVDF membranes were blocked with 5% non-flat milk (Sangon, Shanghai, China) and followed by probing with the corresponding antibodies for CYPA (final concentration: 1 µg/mL; Abcam), p-Stat3 (final concentration: 12 ng/mL; Cell signaling), T-Stat3 (final concentration: 24 ng/mL; Cell signaling), Vimentin (final concentration: 0.27 µg/mL; Proteintech), Cytokeratin 18 (CK18, final concentration: 0.3 µg/mL; Proteintech), β-Tubulin (final concentration: 0.1 µg/mL; Proteintech) and Gapdh (final concentration: 0.2 µg/mL; Santa Cruz) overnight at 4°C. After washing, the matched secondary antibodies conjugated with HRP (final concentration: 0.1 µg/mL; Elabscience) were incubated PVDF membranes. Signals were developed with the ECL kit (Sangon, Shanghai, China).
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