FACS sorted CML LSK cells were treated as described in the results or directly lysed in buffer containing 0.5% Nonidet P-40 (Sigma Diagnostics) and 0.5% sodium deoxycholate supplemented with phenylmethylsulfonyl fluoride (1 mM/L), protease inhibitor mixture, and phosphatase inhibitors (50 mM/L sodium fluoride, 1 mM/L sodium vanadate; all from Sigma Diagnostics). Proteins were resolved on 4% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membrane. Primary antibodies used were anti–EZH1 (cat: ab176115; Abcam, Cambridge, MA), anti–EZH2 (cat: 39933; Active Motif, Carlsbad, CA), anti– H3K27me3 (cat: ab6002; Abcam, Cambridge, MA), anti-actin (clone: AC15; cat: A5441; Sigma Aldrich, St. Louis, MO), anti-H3 (cat: ab1791; Abcam, Cambridge, MA), anti-P-ERK1/2 (Thr202/Tyr204) (cat: 4370S; Cell Signaling, Danvers, MA), anti-P-AKT (Ser473) (cat: 4060S; Cell Signaling, Danvers, MA), and Horseradish peroxidase– conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibody detection was performed by using the SuperPico and SuperFemto kits (Pierce Biotechnology, Rockford, IL).
Anti actin clone ac 15
Manufactured by Merck Group
Anti-actin (clone AC-15) is a mouse monoclonal antibody that recognizes the actin protein. Actin is a ubiquitous and highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used to detect actin in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 1, by Cell Signaling Technology (2 mentions)
Mouse monoclonal anti vimentin clone v9, by Merck Group (2 mentions)
Mouse monoclonal anti nlrp3, by Adipogen (2 mentions)
Anti p erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti ezh2, by Active Motif (1 mentions)
Anti ezh1, by Abcam (1 mentions)
Anti h3k27me3, by Abcam (1 mentions)
Anti h3, by Abcam (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Hrp conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
X ray film, by Fujifilm (1 mentions)
Hybond p membrane, by Cytiva (1 mentions)
Peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Ecl chemiluminescent detection system, by Cytiva (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti irf3, by Active Motif (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
10 protocols using anti actin clone ac 15
1
Protein Extraction and Analysis of CML LSK Cells
2
Analyzing NF-κB Activation in mMSCs
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We determined the activation of NF-κB by using a luciferase reporter system [29 (link)]. Replication incompetent lentiviral vectors were created using the pHAGE NF-κB-TA-LUC-UBC-GFP-W plasmid, a gift from Darrell Kotton (Addgene plasmid # 49343). The plasmid encodes the NF-κB consensus binding sequence upstream of the minimal TA promoter of the herpes simplex virus followed by the firefly luciferase gene, as well as the GFP gene upstream of the ubiquitin-C promoter. Transduction of mMSCs was performed overnight and GFP expression was assessed by flow cytometry. For luciferase-reporter assays, cells were infected with ICOVIR5 and, 24 hours later, 104 cells were lysed and luciferase activity was assayed with the luciferase assay system (Promega), according to the manufacturer's instructions. Activation of Akt and c-Jun was analyzed in the lysates of these cells by western blotting using the following mouse monoclonal antibodies: anti-phospho-Akt-Ser473 (clone 2118, Epitomics), anti-phospho-c-Jun (clone KM-1, Santa Cruz Biotechnology) and anti-Actin (clone AC-15, Sigma-Aldrich).
3
Western Blot Analysis of NS1 Protein
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Cell lysates were loaded onto 10% SDS-PAGE and then blotted on a nitrocellulose membrane. Blots were blocked in PBS with 0.01% Tween-20 and 5% dry milk and stained with anti-NS1 antibodies (clone 4C4 or 29F10, BioSan, Novosibirsk, Russia) or anti-actin (clone AC-15, Sigma) in blocking buffer followed by incubation with secondary HRP-conjugated anti-mouse antibodies (Jackson ImmunoResearch, Cambridge, UK). Blots were developed with ECL reagent (Amersham, Cytia). Signals were registered onto X-ray film (FujiFilm) or by ChemiDoc. Images were analyzed by ImageJ software (https://imagej.nih.gov/ij/ ).
4
Macrophage Inflammasome Activation Assay
5
Macrophage Response to LPS and ATP
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Primary murine macrophages were cultured overnight in 1% serum and treated or not with LPS (100 ng ml−1) for 7 h. ATP (50 μM) was added to LPS-stimulated cells during the last 45 min. Cells were lysed in sample buffer and equal volumes loaded in 10% SDS–PAGE gels transferred to nitrocellulose membranes. The presence of indicated proteins in cell lysates and medium was assessed by western blotting using the following antibodies: mouse monoclonal anti-vimentin (clone V9; 1:500; Sigma-Aldrich no. V 6630, St Louis, MO), mouse monoclonal anti-NLRP3 (1:1,200, Adipogen International no. AG-20B-0014, San Diego, CA), chicken polyclonal anti-vimentin (1:500, Covance no. PCK-594P, Princeton, NJ), anti-pro-IL-1β, anti-IL-1β (clone p34; 1:1,000, Cell Signaling, Danvers, MA), anti-caspase-1 (1:1,000, Cell Signaling, no. 2225, Danvers, MA), and anti-actin (clone AC-15, 1:2,500, Sigma-Aldrich, St Louis, MO). Ponceau S staining was used to assess the release of total protein from activated macrophages. Membranes were blotted with antibodies as described in each figure. Untransfected J774.1 cells and two independent J774.1 clones expressing shRNAs against vimentin were primed with LPS (100 ng ml−1) for 5.5 h and then stimulated with ATP (1 mM) for 25, 50 or 85 min. Control cells (0) were incubated without ATP for 85 min.
6
Quantitative Analysis of Protein and Gene Expression
7
Western Blotting Quantification of hSOD1
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Protein samples were resolved on SDS–polyacrylamide gels and transferred to Hybond-P membranes (Amersham, Pittsburgh, PA). Membranes were blocked for 1 h in TBS, 0.1% Tween-20 and 5% BSA, followed by an overnight incubation with primary antibody diluted in the same buffer. After washing with 0.1% Tween in TBS, the membranes were incubated with peroxidase-conjugated secondary antibody (Amersham) for 1 h, and then washed and developed using the ECL chemiluminescent detection system (Amersham). A human specific rabbit anti-hSOD1 (clone EPR1726, Epitomics, CA) was used to probe for hSOD1. Densitometric analyses were performed using the NIH Image software and normalized against the signal obtained by re-probing the membranes with anti-actin (clone AC-15, Sigma-Aldrich).
8
Immunoblot Analysis of Signaling Pathways
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The procedures used for preparation of Whole Cell Extracts (WCE), resolution by SDS-PAGE electrophoresis and immunoblot was fully detailed in93 . The following primary antibodies were used in this study: anti-MKP-1/DUSP1 (M18, #sc-1102), anti-α-tubulin (B-7, #sc-5286) and anti-NFκB p65 (C-20, #sc-372) were obtained from Santa Cruz Biotechnology. Anti-phosphoT183/Y185 SAPK/JNK (G9, #9255), anti-SAPK/JNK (#9252), anti-phosphoT180/Y182 p38 MAPK (D3F9, #4511), anti-p38 MAPK (D13E1, #8690), anti-phosphoS536 NF-kappaB p65 (93H1, #3033), anti-phosphoS32 IκBα (14D4, #2859), anti-IκBα (#9242), anti-phosphoT71 ATF-2 (11G2, #5112), anti-ATF-2 (20F1, #9226), anti-phosphoS73 c-Jun (D47G9, #3270), anti-c-Jun (60A8, #9165), anti-cleaved Caspase 9 (D315, #9505) and anti-cleaved Caspase 3 (D175, #9664) were from Cell Signaling. Anti-actin clone AC-15 (#A5441) and anti-Flag M2 (#F1804) were purchased from Sigma-Aldrich, anti-IRF3 (#39033) was from Active Motif and anti-RSV (#AB1128) was from Chemicon International. Anti-phosphoS396 IRF3 was described in38 (link) and anti-SeV was obtained from Dr. J. Hiscott, McGill University, Montreal, Canada. HRP-conjugated goat anti-rabbit and rabbit anti-goat (Jackson Laboratories), and goat anti-mouse (Kirkegaard & Perry Laboratories) were used as secondary antibodies. Immunoblots were quantified using the ImageQuantTL software (Molecular Dynamics).
9
Histone Modifications and Protein Expression Analysis
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Cells were lysed for Western blotting in RIPA buffer (Cell Signaling Technology) with complete protease inhibitor cocktail (Roche). For the detection of histone H3 acetylation levels, cells were lysed in SDS lysis buffer (62.5 mM Tris-HCl, 2% SDS, 10% (v/v) glycerol, 1 mM DTT and complete protease inhibitor cocktail). The following antibodies were used: anti-MYCN (B8.4.B, sc-53,993, Santa Cruz), anti-ELOVL2 (EPR11880, ab176327, Abcam), anti-monoubiquitin H2A (Lys119) (ABE569, Millipore), anti-BMI1 (6964, Cell Signaling Technology), anti-RING1A (2820, Cell Signaling Technology), anti-RING1B (5694, Cell Signaling Technology), anti-SREBP1 (sc-13,551, Santa Cruz Biotechnology), and anti-ß-actin (clone AC-15, Sigma-Aldrich).
10
Western Blot Antibody Reagents
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Mouse monoclonal anti-flag (M2 clone; #F1804) and anti-ß-actin (clone AC-15; #A1978) were purchased from Sigma (Castle Hill, NSW). Rabbit polyclonal anti-Mcl-1 (#600-401-394) was purchased from Rockland (Jomar Bioscience, Welland, SA). Hamster monoclonal anti-Bcl-2 (clone 3F11) was obtained from Andreas Strasser (WEHI). Anti-Bcl-w and anti-Bcl-xL were obtained from Lorraine O'Reilly (WEHI) and rat anti-A1 (clone 6D6) was provided by David Huang (WEHI). Cycloheximide (#C7698), 4-hydroxytamoxifen (4HT; #H7904), roscovitine (#R7772) and MG132 (#C2211) were purchased from Sigma.
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