The total cell protein was extracted using lysis buffer (P0013; Beyotime Biosciences, Shanghai, China). We added a mixture of protease and phosphatase inhibitors (B14002 and B15002; Biotool, Shanghai, China), four times the volume of the cell pellet, to the cell lysate. The total protein (35 μg) was separated via SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD-Millipore, Billerica, MA, USA). The membrane was blocked with 5% skimmed milk (232,100; BD Biosciences, Franklin Lakes, NJ, USA) for at least 2 h and then incubated with the appropriate primary antibody overnight at 4 °C. Peroxidase-conjugated secondary antibody was added and incubated at 37 °C for 1 h. The following primary antibodies were used: Anti-ACAT1 (16215-AP-1, 1:1000), anti-P62/SQSTM1 (66184-1-lg, 1:1000), anti-NRF2 (16396-1-AP, 1:1000), anti-lamin B1 (66095-1-lg, 1:1000), anti-FUS (60160-1-lg, 1:1000), and anti-GAPDH (60004-1-Ig, 1:1000) from ProteinTech Group, and anti-LC3B (3868 s, 1:1000) from Cell Signaling Technology (Denver, MA, USA). The total protein was visualized using an enhanced chemiluminescence method (34,080; Thermo Fisher Scientific, Waltham, MA, USA). Image J software (National Institutes of Health, Bethesda, MD, USA) was used to evaluate each band quantitatively and Prism 5.0 software (GraphPad, La Jolla, CA, USA) was used to compare the signal intensity.
Enhanced chemiluminescence method
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Enhanced chemiluminescence method is a laboratory technique used to detect and quantify proteins or other biomolecules in a sample. It relies on the emission of light when a chemical reaction occurs, which is then measured and analyzed to provide information about the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Image lab software v 4, by Bio-Rad (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Image station 4000r, by Kodak (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Anti gapdh, by Proteintech (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ab6161, by Abcam (2 mentions)
Km8009, by Abcam (2 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Non fat milk, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gapdh antibody, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
44 protocols using enhanced chemiluminescence method
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Protein Expression
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For western blot analysis, the cellular extracts were solubilized in 3× Laemmli buffer consisting of 1% SDS and boiled for 5 min at 95 °C. Equal protein amounts (30–80 μg) were separated on SDS–polyacrylamide gel electrophoresis (12.5–15% gels) and transferred to nitrocellulose membranes. Antibody detection was accomplished using the enhanced chemiluminescence method (Thermo Fisher Scientific, Darmstadt, Germany) and developed either with the Fusion SL Imager (Vilber Lourmat, Eberhardzell, Germany) or the Curix60 processor (Agfa healthcare, Bonn, Germany). The following antibodies were used in 3% milk/TBS–Tween (0.1%): anti-mFas (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin (1:40,000, MP Biomedicals, Eschwege, Germany), anti-tubulin (Bio-Rad, Munich, Germany), anti-E-cadherin (BD Transduction laboratories, Heidelberg, Germany), anti-hFas, anti-p65, anti-phospho-p65, anti-IκBα, anti-phospho-IκBα, anti-cleaved caspase-3 and anti-Bcl-xL (Cell Signaling, Leiden, The Netherlands).
3
Protein Expression Analysis of Midbrain Tissues
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Total protein of midbrain tissues and cells was obtained using Invitrogen Lysis buffer and quantified by the BCA method (Thermo Fisher Scientific). An equal amount of protein (50 μg) was subjected to electrophoresis on 10% SDS-polyacrylamide gels and then blotted on nitrocellulose membranes (Thermo Fisher Scientific). After being blocked in 5% non-fat milk, the membranes were probed with anti-KLF4 (ab129473; Abcam) or anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, ab9485; Abcam) antibody, followed by the addition of horseradish peroxidase-conjugated secondary antibody (ab205718; Abcam). Immunoblots were developed by the enhanced chemiluminescence method (Thermo Fisher Scientific) as recommended by the manufacturers.
4
Protein Expression Analysis in Synovium
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The total synovial proteins were extracted using the protein extraction kit (Beibo Corp., Shanghai, China) and then resolved by electrophoresis. Next, the proteins were transferred from the gel onto the PVDF membrane (Millipore Corp., USA) using the semidry method (Tianneng Corp., China). The PVDF membranes were incubated with respective MAPK, PI3K, Akt1,PTEN,p-MAPK, p-PI3K, p-Akt1, and p-PTEN antibodies (dilution 1 : 1000; Abcam Biotechnology, Cambridge, UK) for 2 h at RT, followed by incubation with the following secondary antibodies for 1 h at RT: antimouse secondary antibody (dilution 1 : 5000. Zhongshan Goldenbridge Biotechnology Corp., Beijing, China) for MAPK, PI3K, Akt1, PTEN, p-MAPK, p-PI3K, p-Akt1, and p-PTEN. The enhanced chemiluminescence method (Thermo) was used to develop the bands, and the images were obtained.
5
Whole cell protein extraction and analysis
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The treated cells were lysed in whole cell lysis buffer (62.5 mM Tris-HCl at pH 6.8, 20% glycerol, 2% SDS, 2 mM DTT, 100 µM PMSF, and proteinase inhibitor cocktail). Equal amounts of sample proteins (50 μg) were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, CA, USA). After blocking with 5% nonfat milk, the membrane was probed with designated primary and secondary antibodies and then developed with the enhanced chemiluminescence method (Thermo Scientific) and visualized with the Kodak Image Station 4000R (Kodak, Rochester, USA).
6
Western Blot Analysis of Apoptosis Markers
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Cells were lysed in lysis buffer containing protease inhibitors (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.05 M EDTA, 1% Triton X-100, 0.1%SDS and 0.005×protease inhibitor cocktail). The cell lysates were resolved on 10%–15%SDS-PAGE electrophoresis and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% nonfat milk in PBS-Tween 20 for 1 h at room temperature and incubated with selected primary antibody with β-actin used as an internal control. Immunoreactivity was detected after incubation with a horseradish peroxidase–conjugated secondary antibody (Jackson Immuno Research Laboratories, Inc., PA, USA, 1:2000 dilution) by using the enhanced chemiluminescence method (Thermo Scientific, USA). Antibodies against Bcl-2, B-cell lymphoma-extra large (Bcl-xl), Myeloid cell leukemia-1 (Mcl-1), Bcl-2-associated x protein (Bax), cytochrome c, β-actin (Santa Cruz Biotechnology, CA, USA, 1:1000 dilution) and cleaved caspase 3 p17-specific Antibody (Proteintech, IL, USA, 1:1000 dilution) were used for Western blotting analysis. Western blot band density were analysed using the Image J software per the manufacturer’s instruction, and the band density ratio of each protein to β-actin were calculated accordingly. Three independent experiments were done for final analyses.
7
Quantifying NF-κB p65 Activation
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Whole-cell lysates with approximately 40 μg of proteins were resolved on 10% SDS-PAGE and were subjected to western blot assay using the anti-NF-κB p65 (Cell Signaling Technology, Cat.8242, 1:1000) and anti-phospho-NF-κB p65 (Cell Signaling Technology, Cat.3303, 1:1000). After appropriate secondary antibody incubation, the bands were visualized with the Molecular Imager System (BIO-RAD, Hercules, USA) using an enhanced chemiluminescence method (Thermo Fisher Scientific, Massachusetts, USA)64 (link).
8
Bacterial Modulation of Conjunctival Goblet Cells
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First-passage human conjunctival goblet cells were seeded in six-well plates. After incubation with the three strains of bacteria, supernatant was removed and cells were lysed in RIPA buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% deoxycholic acid, 1% Triton X100, 0.1% SDS and 1 mM EDTA). Protein was collected for analysis by western blot. Briefly, the lysate was centrifuged at 10 000 × g for 10 min at 4°C. Sample buffer was added to the lysate, proteins separated by SDS-PAGE through 10% polyacrylamide and transferred to nitrocellulose membrane to be processed for western blot. The primary antibodies were used for western blots at the following dilutions: 1:200 NLRP3, 1:400 pro-IL1β and 1:1000β-actin. NLRP3 and pro-IL-1β antibodies were purchased from R&D Systems (Minneapolis, Minnesota, USA), and β-actin antibody from Sigma Aldrich (St. Louis). Pro-IL-1β and NLRP3 were normalised to β-actin. For detection, secondary antibodies used were donkey anti-sheep (R&D Systems) and chicken anti-goat (EMD Millipore, Billerica, Massachusetts) conjugated to horseradish peroxidase. Immunoreactive bands were visualised by the Enhanced Chemiluminescence method (Thermo Scientific, Rockford, Illinois, USA). Results were expressed as fold increase above the normalised basal level.
9
Western Blot Analysis Protocol
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Western blot analysis was performed as described previously10 (link),11 (link). Whole-cell lysates with approximately 40 μg of proteins were resolved on 10% and 12% SDS-PAGE and were subjected to western blot assay using the antibodies listed in Supplementary Materials . After appropriate secondary antibody incubation, the bands were visualized with the Molecular Imager System (BIO-RAD, Hercules, USA) using an enhanced chemiluminescence method (Thermo Fisher Scientific).
10
Western Blot Protein Expression Analysis
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After all the designated treatments, cells were collected and rinsed with chilled PBS, then lysed by the Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 2 mM DTT, phosphatase inhibitor and proteinase inhibitor cocktail). Cell lysates were boiled for 10 min. An equal amount of protein was resolved by SDS-PAGE gel and transferred onto PVDF membrane (Bio-Rad, 162–0177). Membranes were blocked in blocking buffer (Thermo Scientific, 37538) for 30 min, then followed by incubation with the designated first and second antibodies and finally developed with the enhanced chemiluminescence method (Thermo Scientific, 34076) and visualized by a Kodak Image Station 4000 R (Kodak). The expression of proteins was quantitatively evaluated by ImageJ.
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