The general reagents were purchased from Sigma-Aldrich (UK), unless stated otherwise. RPMI 1640, Lipopolysaccharide (LPS), Penicillin Streptomycin solution were procured from the Sigma-Aldrich. Recombinant mouse IFN cytokine is from eBiosciences, (San Diego, CA). CD11b+ human and Mouse MACS Microbeads and LC Columns are from MiltenyiBiotec. Primary antibodies including rabbit polyclonal NOS-2, rabbit polyclonal CD-206, rabbit polyclonal β-Actin, mouse monoclonal β-Actin are from Santa Cruz biotechnology. Mouse monoclonal Arginase-1 is from BD Biosciences. Rabbit monoclonal pSTAT3, p38MAPK, and pNF-kB p65 are from Cell Signaling Technology. HRP-linked anti-mouse IgG and anti-rabbit IgG are from Cell Signaling Technology. Anti-mouse/human-CD11b (Clone (M1/70)-FITC-conjugated, anti-mouse CD4 (Clone GK1.5)-PE-conjugated, and anti-mouse-CD8a (Clone 53-6.7)-Per CP/cy5.5-conjugated antibodies and their respective isotype control anti-body including FITC Rabbit IgG2bK (Clone RTK4530), PE Rat IgG2bK (Clone RTH4530) and PerCP/Cy5.5 Rat IgG2bK (RTK4530) were procured from Biolegend (Germany). TNFα, IFNγ, and IL-6 ELISA kits were purchased from R&D system (Darmstadt, Germany).
Mouse monoclonal β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse monoclonal β-actin is a primary antibody that binds to the β-actin protein, a ubiquitous cytoskeletal protein found in eukaryotic cells. It is useful for the detection and quantification of β-actin expression in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp linked anti mouse igg, by Cell Signaling Technology (3 mentions)
Rpmi 1640, by Merck Group (3 mentions)
Cd11b human and mouse macs microbeads and lc columns, by Miltenyi Biotec (2 mentions)
Rabbit polyclonal cd 206, by Santa Cruz Biotechnology (2 mentions)
Rabbit polyclonal β actin, by Santa Cruz Biotechnology (2 mentions)
Mouse monoclonal arginase 1, by BD (2 mentions)
Percp cy5.5 rat igg2bk rtk4530, by BioLegend (2 mentions)
Recombinant mouse ifnγ cytokine, by Thermo Fisher Scientific (2 mentions)
Pnf kb p65, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Il 6 elisa kit, by R&D Systems (1 mentions)
Cd206, by Santa Cruz Biotechnology (1 mentions)
Ciap 1, by Santa Cruz Biotechnology (1 mentions)
Lamp2, by Santa Cruz Biotechnology (1 mentions)
Cd11b human and mouse macs microbeads, by Miltenyi Biotec (1 mentions)
Ciap2, by Santa Cruz Biotechnology (1 mentions)
Ym 1 antibody, by STEMCELL (1 mentions)
Fizz 1 antibody, by Abcam (1 mentions)
Tnfα and il 10 elisa kits, by R&D Systems (1 mentions)
Gentamicin, by Merck Group (1 mentions)
12 protocols using mouse monoclonal β actin
1
Immunologic Characterization of Macrophage Activation
2
Characterization of Murine Macrophage Polarization
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The general reagents were purchased from Sigma-Aldrich (UK), unless stated otherwise. RPMI 1640, lipopolysaccharide (LPS), Gentamicin, MTT and NaNO2, sodium nitroprusside (SNP), penicillin–streptomycin solution, and metformin were procured from Sigma-Aldrich. Recombinant mouse IFNγ cytokine is from eBiosciences (San Diego, CA, USA). CD11b+ human and Mouse MACS Microbeads and LC Columns are from Miltenyi Biotec. Primary antibodies including rabbit polyclonal NOS-2, rabbit polyclonal xIAP, cIAP-1, cIAP-2, LAMP-2, CD-206, rabbit polyclonal β-actin, and mouse monoclonal β-actin are from Santa Cruz biotechnology. Mouse monoclonal arginase-1 is from BD Biosciences. Rabbit monoclonal STAT1 and 3, pp38MAPK, and pNF-kB p65 are from Cell Signalling Technology. Ym-1 antibody was purchased from Stem cell technologies; Fizz-1 antibody was from Abcam. HRP-linked anti-mouse IgG and anti-rabbit IgG are from Cell Signalling Technology. TNFα and IL-10 ELISA kits were purchased from R&D system (Darmstadt, Germany).
3
Macrophage Polarization Pathway Profiling
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All reagents were purchased from Sigma-Aldrich (UK), unless stated otherwise. RPMI 1640, penicillin streptomycin solutions were procured from Sigma-Aldrich. Recombinant mouse IFN-γ cytokine was purchased from eBiosciences (San Diego, CA). CD11b+ human and mouse MACS MicroBeads and LC Columns were purchased from Miltenyi Biotec. Primary antibodies including rabbit polyclonal iNOS, rabbit polyclonal CD-206, rabbit polyclonal, and mouse monoclonal β-actin were purchased from Santa Cruz Biotechnology. Rabbit monoclonal STAT3, pp38MAPK, pNF-kB, and Sphk-1 antibody were purchased from Santa Cruz. S-1PR1 and S-1PR3, HRP-linked anti-mouse IgG, and anti-rabbit IgG were purchased from Cell Signaling Technology. Anti-mouse/human-CD11b (CloneM1/70)-FITC-conjugated antibodies and their respective isotype control antibody including FITC rabbit IgG2bK (Clone RTK4530), PE Rat IgG2bK (Clone RTH4530), and PerCP/Cy5.5 rat IgG2bK (RTK4530) were purchased from Biolegend (Germany). Alexa fluor-488– and Alexa fluor 569–conjugated antibodies were purchased from Invitrogen. IFN-γ and IL-6 ELISA kits were purchased from R&D Systems (Darmstadt, Germany).
4
Western Blot Analysis of Protein Targets
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Cells were harvested and lysed with protease inhibitor-containing EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl, 0.5% NP-40). After lysing, cells were centrifuged at 4 °C. The resulting supernatant was removed prior to quantification of protein concentration using Bradford Assay. Laemmli buffer and β-Mercaptoethanol was added to equal amounts of protein in cell lysates. Protein samples of 100 – 200 μg were subsequently separated with 12% and 8% Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis before transfer to PVDF membrane (Bio-Rad). Primary antibodies were added overnight following blocking in 5% milk in PBS-T, and were as follows: rabbit His-tag antibody (Cell Signalling Technology, RRID:AB_2115720), mouse CHD antibody (Santa Cruz Biotechnology, RRID:AB_10610044), mouse monoclonal p16INK4a antibody (BD Pharmingen, RRID:AB_394077), mouse monoclonal β-actin (Santa Cruz Biotechnology, RRID:AB_2833259), mouse FLAG-tag antibody (Sigma-Aldrich) and rabbit cleaved caspase-3 (CC3) antibody (Cell Signalling Technology). Visualisation was conducted using iBright1500 (Thermo Fisher). Bands were analysed using ImageJ software and normalized to β-actin.
5
Comprehensive Antibody Catalog for Protein Analysis
6
Western Blot Analysis of Cellular Protein
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Protein were extracted from cells or tissues using RIPA lysis buffer. Proteins were then separated by SDS-PAGE. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membrane. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were then incubated with rabbit polyclonal notch3 antibody (ab23426; Abcam, Cambridge, UK), rabbit polyclonal caspase-3 (#9662; Cell Signaling Technology, Beverly, USA), mouse monoclonal Bcl-2 (#15071; Cell Signaling Technology) or mouse monoclonal β-actin (sc-47778; Santa Cruz) overnight at 4°C. The membranes were then incubated 2 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse (Santa Cruz, Dalla, USA) secondary antibody and visualized with a chemiluminescence kit (Pierce, Rockford, USA).
7
Protein Expression Analysis in Cellular Differentiation
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Western blot analysis was performed as described previously [16 (link)] using the following antibodies against rabbit polyclonal Bcl-2 (Sc-492; 1:500), rabbit polyclonal cleaved caspase-3 (Sc-22171-R; 1:400), rabbit polyclonal Bax (Sc-493; 1:500), rabbit polyclonal pAkT (sc–135650; 1:1000), rabbit polyclonal p-PI3-kinase (sc-12929; 1:500) and mouse monoclonal β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The blots were then incubated for 1 h at room temperature with goat anti-mouse secondary antibody (Sc-2005; 1:2000; Santa Cruz Biotechnology) or polyclonal goat anti-rabbit secondary antibody (Sc-66931; 1:5000; Santa Cruz Biotechnology).
To verify if the U937-PMA driven differentiation was also associated with the expression of macrophage-selective markers, we performed Western blot analyses with the following monoclonal antibodies: anti-CD206 (monoclonal, 1:1000, Santa Cruz), anti-CD14 (monoclonal, Santa Cruz). The differentiation of SH-SY5Y cells was detected using the following antibodies: anti-Nestin (monoclonal, 1:1000, Santa Cruz), rabbit anti-MAP2 (policlonal, 1:1000, Millipore) (Figure S1 ).
To verify if the U937-PMA driven differentiation was also associated with the expression of macrophage-selective markers, we performed Western blot analyses with the following monoclonal antibodies: anti-CD206 (monoclonal, 1:1000, Santa Cruz), anti-CD14 (monoclonal, Santa Cruz). The differentiation of SH-SY5Y cells was detected using the following antibodies: anti-Nestin (monoclonal, 1:1000, Santa Cruz), rabbit anti-MAP2 (policlonal, 1:1000, Millipore) (
8
IGF2 and Apoptosis Protein Expression
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Protein were extracted from cells or tissues using RIPA lysis buffer. Proteins were then separated by 12% SDS-PAGE. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membrane. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were then incubated with rabbit polyclonal IGF2 antibody (sc-5622; Santa Cruz, Dallas, USA), rabbit polyclonal antibody (#9662; Cell Signaling Technology, Beverly, USA), rabbit polyclonal antibody (#9504, Cell Signaling Technology), or mouse monoclonal β-actin (sc-47778; Santa Cruz) overnight at 4°C. The membranes were then incubated 2 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse (Santa Cruz) secondary antibody and visualized with a chemiluminescence kit (Pierce, Rockford, USA).
9
Immunoblotting Analysis of Apoptosis Regulators
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Cells were harvested in EBC buffer (50 mM Tris at pH 8.0, 120 mM NaCl, 0.5% NP-40) containing protease inhibitors (Roche). Cell lysates were lysed and quantified by Bradford assay to obtain equal amount of protein extracts, then loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto a polyvinylidene difluoride (PDVF) membrane (Bio-Rad). Membrane blot were incubated with specific primary antibodies as follows: Rabbit polyclonal anti-KIF1Bβ was a gift from Dr. Susanne Schlisio. Mouse monoclonal anti-FLAG, rabbit monoclonal cleaved caspase-3, rabbit monoclonal PARP, rabbit polyclonal caspase-9 were purchased from Cell Signaling Technology. Mouse monoclonal β-actin and rabbit polyclonal GAPDH was purchased from Santa Cruz Biotechnology. Primary antibody signals were detected using either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology).
10
Dimethyl Fumarate and Oxidative Stress
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Dimethyl fumarate (DMF), lypopolysaccharide (from E. Coli serotype 0127:B8) and all other reagents were purchased from Sigma Aldrich (France) unless otherwise specified. CORM-401 and ethyl prop-2-yn-1-yl fumarate (EPF) were synthesized in our laboratories as previously described [26] (link), [27] (link), [28] (link), [30] (link). RPMI-1640 medium, fetal bovine serum (FBS) and L -glutamine were from Lonza, while penicillin, streptomycin and Dulbecco Phosphate Buffer Solution (DPBS) were purchased from Life Technologies (Aubin, France). Antibodies were purchased from Enzo Life Sciences (HO-1 rabbit polyclonal), Cell Signaling Technology (β-actin mouse monoclonal) and Santa Cruz Biotechnology (Nrf2 clone C-20 rabbit polyclonal and Lamin A/C clone N-18 goat polyclonal). Nuclear Extract Kit for the isolation and preparation of nuclear extracts was from Active Motif (Paris, France). The CO sensitive probe COP-1 was kindly provided by Prof. Christopher Chang from the University of California, Berkeley [31] (link).
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