Cells were treated as indicated in individual Fig. legends and whole cell extracts (WCE) were prepared in modified RIPA buffer (Sigma) with addition of protease and phosphatase inhibitors (Roche). Western analysis was performed and quantitated as described (Riggs et al., 2006 (link)). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Dual color precision protein MW markers (BioRad) were separated in parallel. Antibodies were purchased as follows: ERα (D-12, sc-8005), ERβ (sc-53494), Pdcd4 (sc-130545), from Santa Cruz Biotechnology (Santa Cruz, CA); AR (#3202) from Cell Signaling Technology; β-actin from Sigma; and α-tubulin (MS-581-P1) from Thermo Scientific/Lab Vision (Fremont, CA). Chemiluminescent bands were visualized on a Carestream Imager using Carestream Molecular Imaging software (New Haven, CT).
Modified ripa buffer
Manufactured by Merck Group
Sourced in United States
Modified RIPA buffer is a lysis buffer used for the extraction and solubilization of proteins from cells and tissues. It is a commonly used buffer in various biochemical and molecular biology applications, such as Western blotting, immunoprecipitation, and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor, by Roche (2 mentions)
β actin, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bradford reagent, by Thermo Fisher Scientific (2 mentions)
Skimmed milk, by HiMedia (2 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Dual color precision protein mw markers, by Bio-Rad (1 mentions)
Ar 3202, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Western blot luminol reagent, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
Phospho specific akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Smad7, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti il 17, by Abcam (1 mentions)
Anti jak2, by Abcam (1 mentions)
Anti stat3, by Abcam (1 mentions)
10 protocols using modified ripa buffer
1
Western Blot Analysis of Cellular Proteins
2
Protein Extraction from 3T3-L1 Cells
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At the designated time points, 3T3-L1 cells were washed with PBS and homogenized in a modified RIPA buffer (Sigma, St. Louis, MO, USA) containing a proteinase inhibitor cocktail. Later, lysates were collected by centrifugation at 12,074× g for 20 min at 4 °C. Protein concentration was evaluated by bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL, USA) [49 (link)].
3
Immunoblotting Protein Detection Protocol
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Immunoblotting was performed following protocols as described previously [20 (link)]. Cells were lysed in modified RIPA buffer (Sigma-Aldrich) and protein content was measured using Bradford reagent (Thermo Scientific). The cellular protein lysates were run in denaturing polyacrylamide gels and thereafter transferred to PVDF membrane (Millipore) for blocking with 5% skimmed milk (HiMedia). The blots were then probed or re-probed with specific primary antibodies and detected using enhanced chemi-luminescence (ECL) detection system following the manufacturer's protocol. The primary antibodies used were as follows: anti-p38, anti-JNK, anti-ERK1/2, anti-PARP-1, anti-ATG and anti-LC3B-II. The secondary antibodies were horseradish peroxide-conjugated goat anti-rabbit IgG. Expression was quantitated using ImageJ software.
4
Hepatic Akt Phosphorylation Analysis
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Hepatic Akt phosphorylation was determined at the end of treatment at basal states by western blot analysis as described previously [33 (link)]. Whole tissue lysates from snap-frozen liver pieces were homogenized in modified RIPA buffer (Sigma). Lysates were cleared, and equivalent amounts of protein were used as determined by the Bradford (Bio-Rad) assay, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were detected with phospho-specific Akt (Cell Signaling, USA, #9271); β-actin (Abcam, UK, sc-47778) was used as a loading control. All membranes were developed with horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratories, USA) and Western Blot-Luminol Reagent (Santa Cruz Biotechnology, USA). Western blot films were scanned and quantified using Total Lab (Phoenix, USA) software.
5
Immunoblotting Analysis of EMT and Autophagy
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Immunoblotting was performed following protocols as described previously [46 (link)]. Cells were lysed in modified RIPA buffer (Sigma-Aldrich) and protein content was measured using Bradford reagent (Thermo Scientific). The cellular protein lysates were run in denaturing polyacrylamide gels and thereafter transferred to PVDF membrane (Thermo Fisher Scientific, #88518) for blocking with 5% skimmed milk (HiMedia). The blots were then probed or re-probed with specific primary antibodies and detected using enhanced chemi-luminescence (ECL; Thermo Fisher Scientific, #3210) detection system following the manufacturer’s protocol. The primary antibodies used were as follows: anti-E-cadherin (CST, 24E10), N-cadherin (CST, D4R1H), Vimentin (CST, D21H3), β-catenin (CST, D10A8), ATG5 (CST, D1G9), LC3-II (CST, D11), phospho-total Smad-2/3 (CST) and Smad-7 (Santacruz, #sc-365846). The secondary antibodies were horseradish peroxide-conjugated goat anti-rabbit IgG. Expression was quantitated using ImageJ and analyzed through Graph-pad Prism software [47 (link)].
6
Western Blotting of SIJ Samples
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At 24 weeks, another batch of SIJs was harvested and exposed to a mixture of
phosphatase and protease inhibitors (Roche) as well as modified RIPA buffer
(Sigma). Western blotting was then carried out as previously described. Primary
antibodies were purchased as follows: Anti IL-17 (1:1000, Abcam), Anti JAK2
(1:1000, Abcam), and Anti-STAT3 (1:1000 Abcam) and Anti-GAPDH (1:1000 dilution,
Abcam). Anti-rabbit secondary antibodies (Santa Cruz, California) were used. The
resultant images were viewed using enhanced chemiluminescence (Google
Biotech).
phosphatase and protease inhibitors (Roche) as well as modified RIPA buffer
(Sigma). Western blotting was then carried out as previously described. Primary
antibodies were purchased as follows: Anti IL-17 (1:1000, Abcam), Anti JAK2
(1:1000, Abcam), and Anti-STAT3 (1:1000 Abcam) and Anti-GAPDH (1:1000 dilution,
Abcam). Anti-rabbit secondary antibodies (Santa Cruz, California) were used. The
resultant images were viewed using enhanced chemiluminescence (Google
Biotech).
7
Detecting Protein Expression Changes
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Following bacterial infection or stimulation with TLR ligands cells were washed with PBS. Whole protein was extracted with modified RIPA buffer (Millipore, Billerica, MA, USA) containing 1-fold Protease inhibitor cocktail and 1 mM Na3VO4. Subsequently, lysates were centrifuged for 10 min at 18,000 × g. Whole protein content was determined with the Lowry method (DC Protein Assay, BioRad, München, Germany) according to the manufacturer’s instructions. Protein samples were spiked with loading buffer and sample reducing agent (both Invitrogen) and equal amounts of protein were subjected to electrophoresis (MOPS running buffer, Invitrogen, 200 V). Proteins were separated on Bis Tris NuPage® gels (Invitrogen) and transferred onto nitrocellulose membranes using standard conditions. The primary antibodies recognizing IκBζ (1:1,000 dilution) and β-actin (Sigma-Aldrich, Steinheim, Germany; 1:10,000 dilution), respectively, were detected using anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (both from Millipore, Temecula, CA, USA; 1:5,000 dilution) and the appropriate substrate (Immobilon Western Kit; Millipore, Billerica, MA, USA).
8
Brain Protein Expression Analysis
9
Protein Expression Analysis Protocol
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Whole-cell lysates were prepared in modified RIPA buffer (Millipore, Burlington MA, USA) containing 1x protease inhibitor cocktail (Roche, Basel, Switzerland). Total protein concentrations were measured using Bio-Rad protein assay dye regent (Bio-Rad, Hercules, CA, USA). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). The membranes were probed with specific antibodies as indicated, and then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were visualized as an estimate of expression level using enhanced chemiluminescence (ECL) reagent (PerkinElmer, Waltham, MA, USA) and a UVP BioSpectrum Imaging System. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin expression was also estimated as a gel loading control.
10
Western Blot and Immunoprecipitation Assay
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Whole cell lysates were prepared in modified RIPA buffer (Millipore) with protease and phosphatase inhibitors (Roche). Equal amounts of lysate were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with primary antibodies (key resources table) overnight at 4 C and with secondary antibodies at room temperature for 1-2 h, followed by chemiluminescent signal detection using ECL Plus (GE Healthcare).
Immunoprecipitation (IP) assay SUM159 cells were lysed in IP lysis buffer (150 mM NaCl, 50 mM HEPES [pH 7.9], 1 mM EDTA, 10% glycerol, 1% IGEPAL, 1 mM PMSF, and 1 3 complete protease inhibitor cocktail), incubated for 20 min on ice, and debris was pelleted by centrifugation at 13,000 rpm at 4 C. Lysates were precleared by incubating with protein G-Sepharose (Amersham Biosciences) for 1 h with rotation. A 50-mg aliquot was removed for input analysis, and 1-mg aliquots of lysates were incubated overnight with rotation at 4 C with 2 mg of the indicated antibody (key resources table). The next day, protein G-Sepharose (20 mL bed volume) was added to the samples and incubated for 4 h at 4 C with rotation. The resulting immunoprecipitate was washed with IP lysis buffer four times for 5 min at 4 C with rotation. Immunoprecipitates were subject to immunoblot assay using antibodies shown in key resources table.
Immunoprecipitation (IP) assay SUM159 cells were lysed in IP lysis buffer (150 mM NaCl, 50 mM HEPES [pH 7.9], 1 mM EDTA, 10% glycerol, 1% IGEPAL, 1 mM PMSF, and 1 3 complete protease inhibitor cocktail), incubated for 20 min on ice, and debris was pelleted by centrifugation at 13,000 rpm at 4 C. Lysates were precleared by incubating with protein G-Sepharose (Amersham Biosciences) for 1 h with rotation. A 50-mg aliquot was removed for input analysis, and 1-mg aliquots of lysates were incubated overnight with rotation at 4 C with 2 mg of the indicated antibody (key resources table). The next day, protein G-Sepharose (20 mL bed volume) was added to the samples and incubated for 4 h at 4 C with rotation. The resulting immunoprecipitate was washed with IP lysis buffer four times for 5 min at 4 C with rotation. Immunoprecipitates were subject to immunoblot assay using antibodies shown in key resources table.
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