We used our own electrophoresis technique to detect the low-molecular-weight peptide 17.1 (1.5 kD). A 16% polyacrylamide gel was run not to the full length of the glass, so that the low-molecular weight compounds did not run out of the gel. As the peptide marker, we used a synthesized pure peptide 17.1, placed in the adjacent lane (see Supplemental Information) The Hsp70, sTNFR1, and 17.1 peptide were conjugated to CNBr-activated Sepharose 4B (GE Healthcare Chicago, Illinois, USA) according to the manufacturer’s protocol. The Hsp70, sTNFR1, 17.1, and 17.0 peptides were adsorbed onto the respective Sepharose 4B column. The column was thoroughly washed with PBS (phosphate buffered saline)/0.5 М NaCl and PBS alone, and then eluted with 0.25 M triethylamine, pH 12. The eluted material was resolved by SDS-PAGE and blotted onto a nitrocellulose membrane. The biotinylated products were visualized by incubating the membrane with streptavidin-conjugated horseradish peroxidase (HRP) and then with an ECL Plus® kit (GE Healthcare Chicago, Illinois, USA). To detect sTNFR1, the blot was incubated with the rabbit anti-TNFR1 antibodies (1:10,000) and a secondary HRP-conjugated anti-rabbit antibody (GE Healthcare Chicago, Illinois, USA; 1:40,000) and then developed with an ECL Plus® kit.
Ecl plus kit
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Germany, Japan, China
The ECL Plus kit is a chemiluminescent detection system used for Western blot analysis. It provides high sensitivity and a wide dynamic range for the detection of proteins.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (16 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
M mlv reverse transcriptase, by Promega (9 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Polyvinylidene fluoride membrane, by Merck Group (8 mentions)
Sybr green master mixture, by Takara Bio (7 mentions)
Pvdf membrane, by GE Healthcare (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Phospho akt s473, by Cell Signaling Technology (4 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (4 mentions)
Total akt, by Cell Signaling Technology (4 mentions)
Nupage sds gel, by Thermo Fisher Scientific (4 mentions)
Nanovue plus spectrophotometer, by GE Healthcare (4 mentions)
Anti gapdh, by Abcam (4 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (4 mentions)
158 protocols using ecl plus kit
1
Electrophoretic Detection of Low-Molecular-Weight Peptide 17.1
2
Western Blot Analysis of VSVG and ICDH
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Western blot analysis was performed as described previously (Shen et al., 2009 (link); Lin et al., 2017 (link)). Samples were resuspended in SDS-Loading buffer and separated in 15% polyacrylamide gel, then transferred onto PVDF membranes (Millipore). The membrane was blocked in 5% (w/v) BSA for 4 h at room temperature, and incubated with primary antibody at 4°C overnight. The primary antibodies were anti-VSVG (sc-365019, Santa Cruz biotechnology, United States), 1:1000 and anti-ICDH, 1:6000. The ICDH antisera were made in our previous study (Xu et al., 2010 (link)). The membrane was washed five times with TBST buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4), and incubated with 1:5,000 dilution of horseradish peroxidase conjugated secondary antibody (Shanghai Genomics) for 4 h at 4°C. Signals were detected using the ECL plus kit (GE Healthcare, Piscataway, NJ, United States) according to the manufacturer’s specified protocol.
3
Investigating TMZ-Induced Stress Response in NK and CD8+ T Cells
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NK cell and CD8 T cell enrichment were performed using the CD56+ CD16+ NK and CD8+ T Cell Isolation Kit (Miltenyi Biotec), respectively. After magnetic cell separation, NK and CD8+ T cells were seeded in 6-plate wells cells at the density of 106 cells/well and treated with 25 μM of TMZ or vehicle (DMSO), for 10 and 30 min, were washed with cold PBS and lysed in a buffer supplemented with protease and phosphatase inhibitors. Membranes with transferred proteins were incubated with the primary antibody anti-pNRF2 (phosphoSer40, 1:5000, Abcam), anti-NRF2 (1:1000, Abcam) or anti-vinculin (1:10,000). The primary antibody incubation was followed by incubation with peroxidase conjugated to the secondary antibody (anti-rabbit, 1:10,000). A chemiluminescence reaction using the ECL Plus kit (GE Healthcare, Chicago, IL, USA) was detected using G: BOX iChemi system (Syngene, Cambridge, UK).
4
Western Blot Analysis of HIF-1α and CXCR4
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Equal amounts (40 µg) of protein lysate were separated by SDS-PAGE (Bio-Rad, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked with TBST containing 5% non-fat dry milk for 1 h and incubated with a mouse monoclonal primary antibody against HIF-1α (1∶300; ab113642, Abcam) or mouse monoclonal anti-CXCR4 (1∶300; ab58176, Abcam) and mouse monoclonal anti-β-actin (1∶1000; Sigma-Aldrich) overnight at 4°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (Santa Cruz, USA) for 2 h. The membranes were again washed three times in TBST, and the proteins were visualized using the enhanced chemiluminescence (ECL) Plus kit (GE Healthcare Bio-Sciences). β-actin was used as a control. The optical density of protein fragments was quantified using Quantity One software (Bio-Rad, USA).
5
Evaluation of AQ Binding to TseF
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The binding capacity of TseF to different 2-alkyl-4(1H)-quinolone (AQ) molecules were evaluated by fat western blotting as reported67 . Briefly, AQs were solubilized in chloroform as a stock solution of 10 mM, and a minimum of 10 μl containing 0.1, 0,2 or 0.5 μM of AQs were spotted onto nitrocellulose (NitroBind, MSI, USA). The dotted membrane was dried at 24 °C for 1 h and the nitrocellulose was then incubated with 3% (w/v) fatty acid-free BSA (A-6003, isolated by cold ethanol precipitation; Sigma) in TBST (10 mM Tris (pH 8.0), 140 mM NaCl and 0.1% (v/v) Tween 20) for 1 h and then placed in a solution containing the His-tagged proteins (TseF or Fur) diluted in TBST (0.5 mg ml−1) at 4 °C overnight with shaking. The membrane was washed with TBST three times for 10 min each and then incubated with anti-His monoclonal antibody (Santa Cruz Biotechnology, USA) diluted at 1:200 in TBST for 1 h at 24 °C. The incubated membrane was then washed three times for 10 min in TBST at 24 °C and then incubated with 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibodies (Shanghai Genomics) for 1 h. Signals were detected using the ECL Plus Kit (GE Healthcare, Piscataway, NJ) following the manufacturer's specified protocol.
6
Cell Wall Protein Extraction and Analysis
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The hP6E7 antibody was evaluated for its binding capacity against gp70, which is present in the cell wall of S. schenckii and S. brasiliensis. The proteins from cell wall were obtained from yeast cells of S. brasiliensis 5110 and S. schenckii grown in Erlenmeyer flasks containing 400 mL of YCG medium (yeast nitrogen–glucose casamino acids) for 7 days with constant agitation at 9 g at 37°C. Then, the yeast cells were collected and incubated with an extraction solution (2 mM DTT, 1 mM PMSF, and 5 mM EDTA in 25 mM Tris/HCl at pH 8.5) at 4°C for 2 h with mild agitation. The supernatant containing the cell surface proteins was collected and concentrated. Five micrograms of cell wall proteins was separated by electrophoresis using a 12% SDS polyacrylamide gel, and subsequently, the proteins were electrotransferred to Hybond ECL nitrocellulose membranes (GE Healthcare). Finally, the membranes were washed and developed using the ECL Plus Kit (GE Healthcare).
7
Receptor Purification and Analysis
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Dot blots were used to analyze the relative solubility of different samples, while western blots and silver stains were used to detect the receptors and analyze their purity as previously described42 (link),43 (link). Samples were loaded into Novex 10% Bis–Tris Gels (Life Technologies, Waltham, MA) according to the manufacturer’s instructions, with the exception that the samples were incubated at room temperature for 10 min as boiling can cause receptor aggregation. After electrophoresis, the samples were transferred to a nitrocellulose membrane, blocked with milk (5% w/v non-fat dried milk in TBST) for 1 h, and incubated with the rho1D4 monoclonal antibody (1:3000 in TBST, 1 h at room temperature or overnight at 4 ºC). A goat anti-mouse HRP-conjugated secondary antibody (Pierce, Rockford, IL) and the ECL-Plus Kit (GE Healthcare, Pittsburgh, PA) were used to visualize the receptors. The SilverXpress kit (Life Technologies) was used according to the manufacturer’s instructions to do total protein stains. All images were captured on a Biorad GelDoc system. ImageJ was used to compare sample intensities and to analyze sample purity.
8
Evaluation of COX-2 Expression
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Whole-cell extracts were prepared in ice-cold lysing buffer (1 ml PBS) was added with 10 µl Triton X-100, 10 µl SDS 10%, 5 µl DTT 1 M, 6 µl PMSF 0.1%, 10 µl aprotinin). Total proteins (50 µg) were separated by electrophoresis in 10% denaturing SDS/polyacrylamide gel, then transferred to Hybond ECL nitrocellulose membrane (GE Healthcare Europe, Milan, Italy). After saturation of non-specific binding sites with 5% non-fat milk in Tris-buffered saline (TBS) 1x-Tween 20 0.05%, the membrane was immunoblotted overnight at 4°C with the primary antibody against COX-2 (1∶250) and subsequently probed with an anti-goat secondary antibody (1∶1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C. The membrane was stripped (Restore Western Blot Stripping buffer, Pierce Biotechnology, Rockford, IL, USA) and re-immunoblotted with anti-actin primary antibody (1∶10000) and then with anti-rabbit secondary antibodies (1∶7500) (Santa Cruz Biotechnology Inc.). The immunoreactive bands were visualized through enhanced chemiluminescence using the ECL-plus kit (GE Healthcare Europe) following the manufacturer's protocol. The bands were quantified by densitometric analysis using Image J 1.43 software. The results were evaluated as relative units determined by normalization of the density of each band to that of the corresponding actin protein band.
9
Protein Expression Analysis in HCC
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The tissues and cell lines of HCC were lysed by an ice-cold RIPA lysis buffer (Beyotime, China) containing a protease inhibitor cocktail (Sigma-Aldrich, USA). The lysates were quantified using BCA methods and equally loaded on SDS-PAGE gels and electroblotted onto polyvinylidene fluoride (PVDF) membranes. After blocking the membranes with 5% skim milk powder, we incubated the membranes with the primary antibodies (1:1000 dilution) against SOX11 (Epitomics, USA), Bcl-2 (Cell Signaling Technology, USA), cleaved caspase-3 (Cell Signaling Technology), cleaved PARP (Cell Signaling Technology), and GAPDH (Cell Signaling Technology) overnight at 4°C. Next, we cultured the membranes with HRP-conjugated secondary antibodies for 2 h at 25°C. Finally, the membranes were visualized using ECL-PLUS/Kit (GE Healthcare, USA) and the protein was quantified using ImageJ software (National Institutes of Health, USA).
10
Western Blot Analysis of Cellular Proteins
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Equal amounts of protein were submitted to electrophoresis on SDS polyacrylamide gels under reducing conditions and the nitrocellulose membrane was blotted with specific antibodies. Polyclonal antibodies against cleaved PARP-1 (sc-56196), CDK6 (sc-56282), GAPDH (sc-32233), and ACTIN (sc-1616) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-histone H2AX (Ser139) (pH2AX) (9718), P53 (2524), and anti-caspase 3 (9665) were purchased from Cell signaling (Danvers, MA, USA) and P21 were obtained from Abcam (Cambridge, MA, USA). The target proteins were analyzed by chemiluminescence using an ECL Plus Kit (GE-Healthcare, Buckinghamshire, England). Quantitative analyses of the optical intensities of protein bands were determined with UN-SCAN-IT graph digitalizing software (Silk scientific, UT, USA) and normalized by actin or GAPDH for protein expression.
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