Vehicle or TQ-treated breast cancer cell lines and tumor tissues obtained from the mouse study were lysed in RIPA lysis buffer (Li et al., 2010 (link)). Nuclear extracts of TQ treated or control cells were prepared using TransAM nuclear extract kit according to the manufacturer’s instructions. Whole cell lysates was resolved on a 10% SDS polyacrylamide gel electrophoresis (SDS–PAGE) gel. The proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad), blocked with Blocking One (Nacalai Tesque, Inc.), and probed with antibodies of interest overnight at 4°C. The blot was washed with tris-buffered saline with 0.1% Tween-20, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence using Western Bright Sirius HRP substrate (Advansta). Images were captured using a ChemiDoc XRS+ imaging system and analyzed using Image LabTM software (Bio-Rad, Hercules, CA, United States) as described previously (Chua et al., 2010 (link)). Densitometric analysis was done using ImageJ software.
Westernbright sirius hrp substrate
Manufactured by Advansta
Sourced in United States, Germany
WesternBright Sirius HRP substrate is a chemiluminescent detection reagent for Western blotting. It is designed to be used with horseradish peroxidase (HRP)-conjugated secondary antibodies to detect and visualize target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Iblot dry blotting system, by Thermo Fisher Scientific (8 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (5 mentions)
Blocking one, by Nacalai Tesque (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
Spectra multicolour broad range, by Thermo Fisher Scientific (4 mentions)
Microchemi chemiluminescence system, by DNR Bio-Imaging Systems (4 mentions)
Image studio lite 5, by LI COR (4 mentions)
Westernbright ecl hrp substrate, by Advansta (4 mentions)
Magicmark xp western protein standard, by Thermo Fisher Scientific (4 mentions)
Chemidoc xrs imaging system, by Bio-Rad (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (2 mentions)
Immunoblot washing solution, by Advansta (2 mentions)
Biorad mini protean 3 cell, by Bio-Rad (2 mentions)
Advanwash, by Advansta (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
36 protocols using westernbright sirius hrp substrate
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Uterine and Brain Proteins
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Mouse uteri or brains were homogenized in 50 mM Tris-HCl buffer saline (pH 7.6) containing 8 M urea and 1% SDS. After centrifugation, resulting supernatant was added to SDS sample buffer. Protein concentrations were determined by Bio-Rad protein assay (Bio-RAD, Hercules, CA). Proteins were separated on a SDS-PAGE and transferred onto PVDF membranes (GE Healthcare, Pittsburgh, PA). These membranes were blocked with 5% BSA or 5% skim milk in 50 mM Tris-HCl-buffered saline containing 0.1% Tween-20 (TBS-T), incubated with given primary antibodies, washed with TBS-T, incubated with HRP-conjugated secondary antibodies and visualized using WesternBright Sirius HRP substrate (Advansta, Menlo Park, CA).
3
Western Blot Protein Analysis
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Protein samples (15 µg) were denatured and separated on sodium dodecyl sulfate-polyacrylamide gels, followed by semi-dry transfer to nitrocellulose membranes (Amersham, GE Healthcare, Chicago, IL). Membranes were blocked with 5% skim milk powder or 5% BSA in Tris-buffered saline. Primary antibodies were diluted in blocking buffer and incubate at 4 °C overnight (Table S1 ). Horse-radish peroxidase (HRP) conjugated secondary antibodies were incubated for 1 h at room temperature. Chemiluminescence was developed using WesternBright Sirius HRP substrate (advansta, San Jose, CA) according to the manufacture’s instruction and visualized using the ChemiDoc MP Imaging system (Bio-Rad, Hercules, CA). For positive controls 40 ng recombinant murine IL-6 or TNFα (Peprotech, Rocky Hill, NJ) were run along with the samples. Total protein was stained with SYPRO Ruby according to the manufacturer’s instructions (Invitrogen, Waltham, MA) and used for normalization. Analysis was performed using ImageLab 6.0.1 (Bio-Rad, Hercules, CA). Images of all blots can be found in Supplemental Material.
4
Protein Electroblotting and Detection
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Proteins were electroblotted onto a low-fluorescent PVDF membrane using a Trans-Blot Turbo transfer cell and the RTA transfer kit (Bio-Rad Laboratories) for 10 min (25 V, 1.3 A, RT) and processed as described [31 (link)]. For detection, the membrane was incubated with Immuno Blue HRP-Substrate (NH DyeAGNOSTICS, Halle, Germany) for 10 min or with WesternBright Sirius HRP-Substrate (Advansta, Menlo Park, CA, USA) for 2 min, washed in Advan washing solution, and the fluorescence or chemoluminescence recorded (ChemiDoc MP CCD camera system, Bio-Rad Laboratories).
5
NLRP3 Inflammasome Western Blot Protocol
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PBMCs cellular lysates were prepared by gentle shaking and applying 10 pulses of sonication (Branson Sonifier 150) in RIPA buffer supplemented with protease inhibitor PhenylMethylSulfonylFluoride 1 mM. Similarly, plasma samples were diluted in RIPA buffer (1:1, v/v) and sonicated as previously described. Protein content was determined using the Bradford method and electrophoresis was carried out in 12% acrylamide SDS–PAGE, loading 60 µg samples. Then, proteins were transferred to PVDF membranes (Biorad, Hercules, CA, USA) and incubated with primary antibody solutions [1:1000] for NLRP3 (D4D8T, Cell Signaling), IL-1β (D2F3B, Cell Signaling), caspase-1 (2225, Cell Signaling), and gasdermin-D (sc-376318, Santa Cruz) quantification. Membranes were then incubated with their respective secondary antibody [1:2000] and immunolabeled proteins were detected by chemiluminescence method using Western Bright Sirius HRP substrate (Advansta, San Jose, CA, USA). Western blot images were finally quantified using Image-Lab software (Biorad, Hercules, CA, USA).
6
Western Blot Analysis of Protein Signaling
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Whole-cell extracts from vehicle or CYM5478- or cisplatin-treated cells were prepared in RIPA lysis buffer. Lysates were then spun at 10,000 rpm for 10 min to remove insoluble material and stored at −80 °C for later use. The protein content in the lysates was measured by Bio-Rad protein assay dye reagent concentrate (Bio-Rad, Des Plaines, IL, USA) and an equal quantity of protein was resolved on a 10% SDS gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan blocking buffer, and probed with rabbit polyclonal antibodies to total STAT3 and mouse monoclonal antibodies against phospho-STAT3 (Tyr 705), Bax, Bcl-xL, and beta-actin (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. The blot was then washed with TBS with 0.1% Tween-20 and exposed to anti-rabbit–horseradish peroxidase (HRP) conjugate and goat anti-mouse HRP (Sigma-Aldrich, St. Louis, MO, USA) antibodies for 1 h, and finally examined by Western Bright Sirius HRP substrate (Advansta). Images were captured using Chemidoc XRS+ imaging system and analyzed using Image Lab™ software (Bio-Rad, Des Plaines, IL, USA).
7
Western Blot Analysis Protocol
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For western blot analysis cells were washed with phosphate-buffered saline (PBS), harvested by using a cell lifter, and lysed in Radioimmunoprecipitation assay (RIPA) buffer with complete Mini EDTA-free protease inhibitor tablets (Roche) and phosphatase inhibitor cocktail PhosSTOP (Roche). The protein concentration was quantified using the BCA Assay (ThermoFischer Scientific) as described earlier [37 (link)]. 20 μg protein lysate were separated by SDS-gel electrophoresis using a NuPAGE™ 4–12% Bis-Tris protein gel and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (all ThermoFischer Scientific). As protein standards, 10 μl Spectra Multicolour Broad Range (ThermoFisher Scientific) and 1 μl MagicMark™ XP Western Protein Standard (ThermoFisher Scientific) were used. For detection, the membranes were incubated with WesternBright Sirius HRP substrate (Advansta). Except for the membranes displayed in S2C Fig , all signals were detected by a Microchemi chemiluminescence system (DNR Bio-Imaging Systems). S2C Fig had been digitalised by using an Odyssey CT (LI-COR). Antibodies used are listed in S2 Table . Densitometric analysis of experiments was made with the Image-Studio Lite 5.2 software (LI-COR). Uncropped western blot images are displayed in the supplementary files. Raw images files are displayed in S1 Raw images .
8
Western Blot Analysis of Nuclear Proteins
9
Gsdmd Protein Expression in BV-2 Cells
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BV-2 cells were treated with vehicle, 200 μM DHA, or 200 μM cisplatin for 2 h. Whole-cell lysates were prepared in RIPA lysis buffer. Lysates were then spun at 10,000 rpm for 10 min to remove insoluble material and protein content in the lysates was measured by Bio-Rad protein assay dye reagent concentrate (Bio-Rad, USA). Lysates were electrophoresed on a 10% SDS gel, electrotransferred to a nitrocellulose membrane, blocked with Blocking One (Nacalai Tesque, Inc., JAPAN) blocking buffer, and probed with a rabbit polyclonal antibody to Gsdmd (Cell Signaling Technologies #93709) overnight at 4 °C. The blot was then washed with TBS with 0.1% Tween-20 and exposed to anti-rabbit–horseradish peroxidase (HRP) conjugate for 1 h, then examined by Western Bright Sirius HRP substrate (Advansta). Three independent experiments were performed and run on the same gel for analysis. Images were captured using Chemidoc XRS + imaging system and analyzed using Image Lab software (Bio-Rad, USA). Band density was quantified with ImageJ.
10
Quantitative Western Blot Analysis Protocol
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As described earlier, subcellular fractionation, cell harvest, protein determination, and western blot were performed [30 (link)]. SDS-gel separated 20 µg protein lysate electrophorese using NuPAGE™ 4–12% Bis-Tris protein gels and transferred to a nitrocellulose membrane using the iBlot dry blotting system (all Thermo Fisher Scientifc, Waltham, MA, USA). Additionally, 5 µL Spectra Multicolour Broad Range (Thermo Fisher Scientifc, Waltham, MA, USA) protein standard and 1 µL MagicMark™ XP western protein standard (Thermo Fisher Scientifc, Waltham, MA, USA) were used. For detection, the membranes were incubated with WesternBright Sirius HRP substrate (Advansta), and signals were detected by a Microchemi chemiluminescence system (DNR Bio-Imaging Systems, Ha-Satat, Israel). The antibodies used are listed in Table 2 . Densitometric analysis of experiments was performed with the Image-Studio Lite 5.2 software (LI-COR, Lincoln, NE, USA). Uncropped western blot images are displayed in the Supplementary Materials . Raw images files are displayed in S1_raw_images.
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