HEK293 cells were transfected with miRNA expression cassettes as indicated. At 48 h cells were rinsed once with iced-cold PBS and lysed with Passive lysis buffer (PBL, Promega). Protein concentration was determined by the Bradford–Lowry method (BioRad) and 10 μg of protein loaded on a NuPAGE 3–8% Tris-Acetate gel (Novex Life Technologies). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with a mouse anti-Htt (1:5000, Millipore, CA, USA), or rabbit anti-Beta-actin (1:40000, Sigma) antibodies followed by horseradish peroxidase-coupled antibodies (1:10,000, mouse; or 1:50,000, Rabbit; Jackson ImmunoResearch, West Grove, PA, USA). Blots were developed with ECL-Plus reagents (Amersham Pharmacia). Silencing efficacy was determined by densitometry (n = 4 independent experiments) of protein levels relative to beta actin with the VersaDocTM Imaging System (Biorad) and Quantity OneR analysis software.
Rabbit anti beta actin
Manufactured by Merck Group
Sourced in United States
Rabbit anti-beta actin is a primary antibody that specifically binds to the beta actin protein, a ubiquitous cytoskeletal protein found in eukaryotic cells. This antibody is commonly used in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and quantify the expression levels of beta actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase coupled antibodies, by Jackson ImmunoResearch (1 mentions)
Versadoc imaging system, by Bio-Rad (1 mentions)
Ecl plus reagent, by Cytiva (1 mentions)
Bradford lowry method, by Bio-Rad (1 mentions)
Phenylmethylsulfonyl fluoride, by Cell Signaling Technology (1 mentions)
Bullet blender storm, by Next Advance (1 mentions)
Secondaryantibody anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions)
Radioimmunoprecipitation assay ripa buffer, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
0.22 μm nitrocellulose membrane, by GE Healthcare (1 mentions)
Goat anti il 1β p17 subunit, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Mouse anti γ tubulin, by Abcam (1 mentions)
Rabbit anti gapdh, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Superblock tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
6 protocols using rabbit anti beta actin
1
Western Blot Analysis of Huntingtin Protein
2
Western Blot Analysis of Protein Expression
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A protein extraction buffer was prepared by adding one protease inhibitors (Sigma, St. Louis, MO, USA) per 10 mL Radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, USA) with phenylmethylsulfonyl fluoride (Cell Signaling Technology, Danvers, MA, USA) at a final concentration of 1 mM. Two volumes of the protein extraction buffer were added to 1 volume of tumor and homogenization was performed with a Bullet Blender Storm (Next Advance, Troy, NY, USA), followed by 10 min centrifugation at 12,000× g at 4 °C. Protein concentration was quantified with BCA protein assay (Thermo Fisher Scientific, Tampa, FL, USA). Under reducing conditions, total protein was fractionated using sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Membranes were probed with rabbit anti-p65 or anti-p100 (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-beta actin (1:1000 dilution, Sigma, St. Louis, MO, USA). Membranes were washed and incubated with secondary antibody anti-rabbit HRP (1:10,000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). Bands were visualized with Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Tampa, FL, USA) using ChemiDoc MP (Bio-Rad Laboratories, Hercules, CA, USA). Knockdown of proteins was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
3
Immunoblotting analysis of inflammasome components
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BMDMs were lysed with a RIPA buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1% (w/v) sodium deoxycholate and 0.1% (w/v) SDS) supplemented with a protease inhibitor cocktail. Precleared lysates and supernatants were boiled with Laemmli buffer, resolved by SDS–polyacrylamide gel electrophoresis and transferred to a 0.22-μm nitrocellulose membrane (GE Healthcare). Rat anti-caspase-1 p20 monoclonal antibody clone 4B4 (Genentech, 1:500), mouse anti-caspase-1 p10 (Santa Cruz Biotechnology, 1:250), goat anti-IL-1β p17 subunit (Sigma-Aldrich, 1:250), rat anti-caspase-11 p10 (Abcam, 1:500) and rabbit anti-beta actin (Sigma-Aldrich, 1:5,000) were used for antigen detection. To detect the expression of Flag-fused effectors by L. pneumophila, bacterial heavy patches streaked from CYE plates after 2 days of growth in the presence of 1 mM IPTG were boiled in Laemmli buffer, immunoblotting was carried out as described above, and anti-Flag M2 (Sigma-Aldrich, 1:5,000) antibody was used for antigen detection.
4
Western Blotting of hPD-L2 in Cultured Cells
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Cultured cells were lysed with lysis buffer and used for Western blotting. The following primary antibodies were used: goat anti-hPD-L2 (R&D Systems) or rabbit anti-beta actin (Sigma-Aldrich, St. Louis, MO), as a control.
5
Western Blotting and Immunoprecipitation Protocol
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For western blotting, mouse anti-Stat3 (Novus Biologicals; 1:1,000), mouse anti-Stathmin (Santa Cruz Biotechnology sc-55531; 1:200), rabbit anti-Stathmin (Novus Biologicals NB110-57602; 1:1,000), mouse anti-α-tubulin (Sigma Aldrich; 1:2,000), rabbit anti-GAPDH (Sigma Aldrich; 1:500), mouse anti-PLK1 (pT210; BD Bioscience; 1:1,000), rabbit anti-PLK1 (Novus Biologicals; 1:500), rabbit anti-Beta-Actin (Sigma Aldrich; 1:5,000) and mouse anti-γ-tubulin (Abcam; 1:1,000) were used. Cells were lysed using 1% NP-40 in Tris-buffered saline. SDS–polyacrylamide gel electrophoresis and western blotting were performed as described previously60 (link). Immunoprecipitations were performed using 1% NP-40 in Tris-buffered saline with added phosphatase inhibitors (sodium vanadate and sodium fluoride) and protease inhibitor (Roche, Basel, Switzerland) and 5 μg mouse anti-Stat3 antibody (Santa Cruz Biotechnology). Uncropped scans of western blots are shown in Supplementary Fig. 6 .
Liver-derived microsome assays and UPLC-UV detection of KM08165 conversion into Stattic was performed by the Centre for Drug Research and Development (Vancouver). In silico prediction of KM08165 substructures was performed with the help of the Centre for Drug Research and Development.
Liver-derived microsome assays and UPLC-UV detection of KM08165 conversion into Stattic was performed by the Centre for Drug Research and Development (Vancouver). In silico prediction of KM08165 substructures was performed with the help of the Centre for Drug Research and Development.
6
Western Blot Analysis of Cell Cycle Regulators
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