Inhibition of the mouse or human CSF-1R was done using the following species-specific blocking antibodies: monoclonal rat anti-mouse CSF-1R (AFS98) or monoclonal mouse anti-human CSF-1R (MAB3291; R&D Systems) (47 (link)) at final concentrations of 50 μg/ml. Transwells were prepared as described above, and blocking antibody or control immunoglobulin G (IgG) was added to the top well containing tumor cells and macrophages.
Mab3291
Manufactured by R&D Systems
MAB3291 is a monoclonal antibody that recognizes human ZBTB17. It is suitable for use in various immunoassay applications, including ELISA and Western blotting.
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Lab products found in correlation
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Horseradish peroxidase linked secondary antibody, by Jackson ImmunoResearch (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Ab107115, by Abcam (2 mentions)
Ab53041, by Abcam (2 mentions)
Anti brdu, by Roche (2 mentions)
Anti krt8 18, by Cell Signaling Technology (2 mentions)
Anti cldn7, by Thermo Fisher Scientific (2 mentions)
H00004950 m03a, by Novus Biologicals (2 mentions)
Anti cleaved caspace 3, by Cell Signaling Technology (2 mentions)
Anti ki67 vp k451, by Vector Laboratories (2 mentions)
Csf1r gene, by Horizon Discovery (2 mentions)
Smad3 gene, by Horizon Discovery (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Signalfire plus ecl reagent, by Cell Signaling Technology (1 mentions)
Ab51253, by Abcam (1 mentions)
Ab138501, by Abcam (1 mentions)
Ab52968, by Abcam (1 mentions)
6 protocols using mab3291
1
Inhibition of CSF-1R in Mice and Humans
2
Protein Expression and Phosphorylation Analysis
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Cells were lysed on ice in a lysis buffer composed of 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) and 5 mM EDTA with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 500 g for 30 min at 4 °C to remove cellular debris. Proteins (25 μg/lane) were separated on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were immunoblotted for CSF1R (1:250; #MAB3291, R&D Systems) and its phosphorylated form (Y723; 1:500; #3155, Cell Signaling), nuclear factor kappa B (NF-κB; 1:500; #8282, Cell Signaling) and its phosphorylated form (S536; 1:500; #3033, Cell Signaling), caspase-1 (1:500; #ab179515, Abcam), IL-1β (1:500; #12,242, Cell Signaling), NLR family pyrin domain containing 3 (NLRP3; 1:500; #15101S, Cell Signaling) and GAPDH (1:5000; G8795, Sigma Aldrich) overnight at 4 °C, and then with horse radish peroxidase-linked secondary antibodies (1:10,000; Jackson Laboratory) for one hour. Bands were detected by enhanced chemiluminescence SignalFire™ Plus ECL reagent (Cell Signaling) using a ChemiDoc Imaging System (Bio-Rad Laboratories). Image analysis was performed using ImageLab 6.0.1 software (Bio-Rad Laboratories).
3
Inhibition of CSF1R in vitro
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Inhibition experiments antibodies: anti-mouse CSF1R antibody (AFS98) 46 (link), anti-human CSF1R antibody (MAB3291, R&D Systems, Minneapolis, MN). Western blot antibodies: anti-KRT8/18 (4546, Cell Signaling, Danvers, MA), anti-CLDN7 (34–9100, Invitrogen), anti-CLDN11 (ab53041, Abcam, Cambridge, MA), anti-OCLN (H00004950-M03A, Novus Biologicals, Littleton, CO). Immunohistochemistry: anti-cleaved caspace-3 (9661, Cell Signaling), anti-Iba1 (019–19741, Wako Chemicals, Richmond, VA), anti-Ki67 (VP-K451, Vector, Burlingame, CA), anti-BrdU (11170376001, Roche, New York, NY) and anti-KRT8 (ab107115, Abcam). siRNAs: CSF1R gene (M-003109-03–0005, Dharmacon, Pittsburgh PA), Smad3 gene (M-020067-00–0005, Dharmacon), both transfected by nucleofection as per the manufacturer’s instructions (Lonza, Basel, Switzerland).
4
Immunoblot Analysis of Protein Expression
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Cells were lysed on ice in a lysis buffer composed of 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) and 5 mM EDTA with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 500g for 30 min at 4°C to remove cellular debris. Proteins (25 μg/lane) were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). For the detection of α-syn, membranes were fixed in 4% paraformaldehyde solution as previously described.38 (link) Membranes were immunoblotted for AXL (AF154 R&D Systems at 1:500), MerTK (ab52968, Abcam at 1:500), phosphorylated MerTK (p186-749, Phosphosolutions at 1:1000), α-syn (ab138501, Abcam 1:5000), S129-phosphorylated α-syn (ab51253, Abcam 1:1000), CSF1R (MAB3291, R&D systems; 1:250) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G8795, Sigma Aldrich at 1:5000) overnight at 4°C, and then with horse radish peroxidase-linked secondary antibodies (1:10 000; Jackson Laboratory) for 1 h. Bands were detected by enhanced chemiluminescence with Clarity Max ECL substrates (Bio-Rad Laboratories) using a ChemiDoc Imaging System (Bio-Rad Laboratories). Image analysis was performed using ImageLab 6.0.1 software (Bio-Rad Laboratories).
5
Inhibition of CSF1R in vitro
6
BT20 Cancer Cell Adhesion, Motility, and Invasion Assays
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The wild-type terminal 578 nt of CSF-1R mRNA 3′UTR (3217-3992 nt; 3′UTR) or the deletional mutant missing the 69 nt pyrimidine-rich sequence (3′UTRΔ69) cDNA was subcloned into pcDNA 3.1 (+) (Invitrogen) [13] (link).
The Membrane Invasion Culture System was used for assay of in vitro adhesion, motility, and invasion of BT20 breast cancer cells. Procedures were followed as described previously, except that the invasion assays were performed for 72 h [4] (link), [14] (link). The degree of BT20 cell adhesion onto a filter coated with human-extracellular matrix consisting of type IV collagen, laminin, and gelatin, was measured at A585 after cell staining. The directed motility assays of BT20 cells were performed using fibronectin (25 μg/ml) as a chemoattractant in the lower wells, above which uncoated 10 μm pore filters were placed. Invasiveness was measured through a filter coated with human extracellular matrix barrier. When the effect of CSF-1R monoclonal antibody was studied, this matrix was pretreated with medium alone, 20 μg/ml mouse idiotype-matched IgG in medium, or 20 μg/ml mouse monoclonal IgG to human CSF-1R (MAB3291, R&D Systems) in medium, each for 1 h. The results are reported as mean A585 for adhesion assay, mean percent motility, or mean percent invasion ± SEM. Three independent experiments were performed.
The Membrane Invasion Culture System was used for assay of in vitro adhesion, motility, and invasion of BT20 breast cancer cells. Procedures were followed as described previously, except that the invasion assays were performed for 72 h [4] (link), [14] (link). The degree of BT20 cell adhesion onto a filter coated with human-extracellular matrix consisting of type IV collagen, laminin, and gelatin, was measured at A585 after cell staining. The directed motility assays of BT20 cells were performed using fibronectin (25 μg/ml) as a chemoattractant in the lower wells, above which uncoated 10 μm pore filters were placed. Invasiveness was measured through a filter coated with human extracellular matrix barrier. When the effect of CSF-1R monoclonal antibody was studied, this matrix was pretreated with medium alone, 20 μg/ml mouse idiotype-matched IgG in medium, or 20 μg/ml mouse monoclonal IgG to human CSF-1R (MAB3291, R&D Systems) in medium, each for 1 h. The results are reported as mean A585 for adhesion assay, mean percent motility, or mean percent invasion ± SEM. Three independent experiments were performed.
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