The kidney tissue samples were lysed using radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology) and the total protein concentrations were detected using the Micro BCA protein assay kit (Guangzhou Youdi Biotechnology Company, Guangzhou, China). Total cell lysates (50 µg) were loaded into each lane and resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to PVDF membranes (Thermo Fisher Scientific, Inc.). The PVDF membranes were blocked with 5% non-fat dry milk at room temperature for 1 h and immunoblotted with anti-KLF15 (ab2647; dilution 1:200) and anti-NF-κB (ab32360; dilution 1:1,000) at 37°C for 2 h, followed by incubation with the secondary antibodies (ab6789; dilution 1:5,000) for another 1 h at room temperature. All antibodies were purchased from Abcam (Cambridge, MA, USA) Visualization was then performed using an enhanced chemiluminescence western blotting detection reagent (Thermo Fisher Scientific, Inc.). Finally, the protein bands were scanned and quantified using a ChemiDoc™ MP Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Enhanced chemiluminescence western blotting detection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced chemiluminescence western blotting detection reagents are laboratory products designed to detect and visualize targeted proteins in western blot analysis. They provide a chemiluminescent signal that can be captured and measured to quantify the presence and relative abundance of specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Microchemi system, by DNR Bio-Imaging Systems (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Dnmt3a, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Dnmt1, by Abcam (1 mentions)
Dnmt3b, by GeneTex (1 mentions)
Histone h3, by GeneTex (1 mentions)
Horseradish peroxidase conjugated donkey anti goat igg, by Abcam (1 mentions)
Cnm compartmental protein extraction kit, by BioChain (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Blocking buffer, by Merck Group (1 mentions)
14 protocols using enhanced chemiluminescence western blotting detection reagent
1
Western Blot Analysis of KLF15 and NF-κB
2
Western Blot Analysis of Primary Chondrocytes
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The proteins from the primary chondrocyte cultures (n = 3 independent isolations) were harvested using 300 µL RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN). The total cell lysates (containing 20–30 µg protein) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corporation, Billerica, MA, USA). The membranes were then blocked using 5% skimmed milk in PBS with 0.25% Tween-20 (PBST) and incubated at 4 °C overnight with the primary antibodies listed in Supplementary Materials Table S2 . After washing with PBST, the membranes were bound to horseradish peroxidase-conjugated secondary antibodies and incubated at RT for 2 h. After washing with PBST, the blots were developed using enhanced chemiluminescence Western blotting detection reagent (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed with the MicroChemi system (DNR Bio-imaging Systems, Neve Yamin, Israel). The Western blot band intensities were quantified with ImageJ software. A list of the primary antibodies used for the Western blot analysis are shown in Supplementary Materials Table S2 .
3
Quantifying Epigenetic Regulators in Rat Lungs
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Lungs were obtained from rats at the end of experiment. Nuclear fraction was collected by using the CNM compartmental protein extraction kit (BioChain Inc., San Francisco, CA, USA) according to the manufacturer’s instruction. Twenty μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membrane was incubated with primary antibody (DNMT1, 1:1000, purchased from Abcam, Cambridge, UK; DNMT3A, 1:300, purchased from Abcam, Cambridge, UK; DNMT3B, 1:1000, purchased from GeneTex, Irvine, CA, USA; Histone H3, 1:5000, purchased from GeneTex, Irvine, CA, USA) at 4°C for 24 h, and then incubated with horseradish peroxidase-conjugated donkey anti-goat IgG (Abcam, Cambridge, UK) or horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell signaling Technology Inc, Danvers, MA, USA) at room temperature for 2 h. The protein levels were detected by the enhanced chemiluminescence Western blotting detection reagent (Thermo scientific, Rockford, IL, USA), and quantified by the ImageJ software version 1.46r (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Protein Expression
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The expression of proteins was measured by western blot analysis. After treatment with Aβ25-35 in the presence or absence of CORT, total protein samples were isolated using radioimmunoprecipitation assay buffer (Sigma-Aldrich). Protein samples were separated in 10% or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Pall Co., MI, USA). The membranes were blocked with 5% nonfat milk in 0.1% Tween 20 in PBS (PBST) for 30 min at RT and then incubated with primary antibodies at 4°C overnight. The dilution factors of primary antibodies were as follows: Bim, Bcl-2, PARP, and Nrf2, 1:500; cleaved caspase-3, COX-2, TNF-α, IL-1β, 4-HNE, pNrf2, GCS, NQO-1, and MnSOD, 1:1,000; Actin, 1:4,000. After washing the primary antibodies, the blots were reacted with horseradish peroxidase-conjugated anti-rabbit (1:10,000; Sigma-Aldrich) or anti-mouse secondary antibody (1:10,000; Santa Cruz Biotechnology). The specific bands were visualized by enhanced chemiluminescence western blotting detection reagent (Thermo, Rockford, IL, USA).
5
Western Blot Analysis of Protein Samples
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Cell lysates were prepared as previously described [54 (link)]. Proteins (30 μg) were resolved on 12% SDS-PAGE and electro-transferred onto PVDF membranes. Those membranes were incubated for 2 h in blocking buffer (Sigma Aldrich), then overnight at 4 °C with specific primary antibodies (0.5 μg/mL). Membranes were washed twice with [50 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% (v/v) Tween-20] (TBST) and incubated for 2 h with appropriate HRP-conjugated secondary antibody (1/20,000). After final washes, the signals were visualized with enhanced chemiluminescence western blotting detection reagent (Thermo Fisher Scientific) on the ChemiDoc XRS+ apparatus (BioRad Laboratories, Marnes-la-Coquette, France) and quantified using the ImageJ software.
6
Ovarian Cancer Cell Line Cultivation
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Four ovarian cancer cell lines, SKOV3, Hey, OVCAR433, and OVCAR5, were used. The SKOV3, OVCAR5 and OVCAR433 cells were grown in DMEM/F12 medium supplemented with 10% bovine serum. The Hey cell line was maintained in RPMI 1640 medium supplemented with 5% bovine serum, 100 units/ml penicillin, and 100 microgram/ml streptomycin under 5% CO2.
SPR965 was obtained from Sphaera Pharma (Singapore). The Annexin V FITC kit was purchased from BioVision (Mountain View, CA). All antibodies were purchased from Cell Signaling (Beverly, MA). Enhanced chemiluminescence western blotting detection reagents were purchased from Thermo (Rockford, IL). All other chemicals were purchased from Sigma (St. Louis, MO).
SPR965 was obtained from Sphaera Pharma (Singapore). The Annexin V FITC kit was purchased from BioVision (Mountain View, CA). All antibodies were purchased from Cell Signaling (Beverly, MA). Enhanced chemiluminescence western blotting detection reagents were purchased from Thermo (Rockford, IL). All other chemicals were purchased from Sigma (St. Louis, MO).
7
Western Blot Analysis of p53 in Irradiated Cells
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24 hours post irradiation, cells were washed once with PBS and scraped and harvested in sample buffer (2.5% w/v SDS, 25% v/v Glycerol, 125mM Tris pH-6.8, 0.01% w/v bromophenol blue, 4% β-mercaptoethanol, in water). Samples were sonicated for ten seconds (30% amplitude, Branson Digital Sonifier) and heated at 95°C for 5 minutes. Proteins were resolved on 10% SDS-polyacrilamide gels and transferred to 0.45μm nitrocellulose membranes (Bio-Rad). Western blots were probed with anti-mouse p53 (Cell signaling) and GAPDH (Millipore). Antibodies were detected using peroxidase-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Labs) and enhanced chemiluminescence western blotting detection reagents (Thermo Fisher scientific). Gels were Imaged by CemiDoc XRS+ (Bio-Rad) and analyzed by ImageLab 5.1 software (BioRad).
8
Western Blot Analysis of CXCL12 Protein
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Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) on ice. Protein concentrations were determined using the BCA kit (Thermo Fisher Scientific, Inc.). The sample proteins (40 µg/lane) were separated via SDS-PAGE on 15% gels and transferred to a PVDF membrane (EMD Millipore). The membranes were then blocked with TBS containing 5% non-fat dried milk and 0.1% Tween-20 at 4°C for 2 h. After blocking, the membranes were incubated with a primary anti-CXCL12 antibody (1:1,000; Abcam; cat. no. ab9797) overnight at 4°C with gentle shaking and subsequently incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:50,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BA1054) for 2 h at room temperature. The bands were detected using enhanced chemiluminescence western blotting detection reagents (Thermo Fisher Scientific, Inc.). GAPDH (1:1,000; cat. no. AB-P-R 001; Goodhere Biological Technology) was used as a loading control, and the band density was measured using ImageJ (version 1.52a; National Institutes of Health).
9
Protein Expression Analysis by Western Blot
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The total proteins were extracted from cells and their concentration was determined using the Pierce™ BCA protein Assay kit (Thermo Fisher). Proteins were separated by 10% SDS–PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk at 37 °C for 1 h. Subsequently, the membranes were incubated with the primary antibodies at 4 °C overnight, followed by incubation with the secondary antibody at 37 °C for 1 h. The target proteins were visualized using Enhanced chemiluminescence Western blotting detection reagents (Thermo Fisher). The antibodies were listed as follows: E-cadherin (Abcam, ab40772), N-cadherin (Abcam, ab18203), vimentin (Abcam, ab137321), GADPH (Abcam, ab8245), CD9 (Abcam, ab92726), and CD81 (Abcam, sc-9158).
10
Femur Decalcification and Protein Extraction
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