Cells were harvested and lysed with RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP40) supplemented with proteinase inhibitor (cOmplete ULTRA Tablets, Mini, Roche). Next, 12.5 μg protein was separated by SDS‐PAGE on 10% polyacrylamide gels and transferred to PVDF membranes. Human anti‐NECTIN4 antibody (1:1000, AF2659; R&D systems) and HRP‐conjugated anti‐goat IgG antibody (1:2000; Dako) were used to detect NECTIN4; anti‐GAPDH (1:2000, MAB374; Millipore) and HRP‐conjugated anti‐mouse IgG (1:1000) were used as loading controls.
Af2659
Manufactured by R&D Systems
Sourced in United States
AF2659 is a laboratory reagent and research tool manufactured by R&D Systems. It is a recombinant human protein that functions as a cytokine. The core function of AF2659 is to facilitate research and analysis in cell biology and biochemistry applications.
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4 protocols using af2659
1
NECTIN4 Protein Expression Analysis
2
NECTIN-4 Protein Expression Analysis
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HT1376 CTRL and polyclonal KO cells were seeded on a 6-well plate. At a confluency of 80% to 90%, cells were collected and lysed in RIPA lysis buffer containing protease inhibitors (#11423129; R-Biopharm, Roche). The Protein concentration of the lysates was determined using the BCA protein assay (#23225, Pierce BCA Protein Assay Kit). A 4X SDS Sample Loading Buffer [Tris-HCl (0,2 mol/L), DTT (0,4 mol/L), SDS (277 mmol/L), 8.0% (w/v) Bromophenol blue (6 mmol/L), Glycerol (4,3 mol/L)] was added and samples were cooked at 95°C for 5 minutes right before loading of the 8% SDS-Gel. After running the Gel proteins were blotted to a 0,2 μmol/L Nitrocellulose membrane (#10600004; AmershamTM ProtranTM Premium). After transfer, the membrane was blocked in 1x Tween-20 TBS containing 5% BSA for 60 minutes. Then, the membrane was incubated with primary antibodies against NECTIN-4 (1:500, AF2659, R&D) and beta-Actin (1:1,000, sc47778, SCB) in TBS containing 2,5% BSA overnight at 4°C. Secondary antibody incubation was performed applying fluorescence labeled anti-mouse and anti-goat antibodies (#926–68072 and #926–32214, LICOR,) for 1 hour in in TBS containing 2,5% BSA. Fluorescence signal was detected using the Odyssey CLx Imaging System (LICOR).
3
Nectin-4 Immunological Detection
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Polyclonal goat antibodies raised against the C-terminal cytoplasmic domain of human nectin-4 was used for immunostaining and western blotting (AF2659; R&D, Minneapolis, MN). In addition, rabbit polyclonal anti-human nectin-4 antibodies were raised against amino acid residues 399–415 (CRRLHSHHTDPRSQPEES) and residues 463–479 (CPGSGRAEEEEDQDEGIK) within the cytoplasmic domain of human nectin-4. The resulting antisera were affinity purified on columns coupled to the peptide. To detect cells of the trophoblast lineage, anti-pancytokeratin monoclonal antibodies (Nichirei, Tokyo, Japan) were used.
4
Nectin-4 Expression in Breast Cancer
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Tissue microarrays were constructed by extracting 2-mm diameter cores of histologically confirmed invasive breast carcinoma areas, as previously described.30 (link) Five-micrometer tissue sections were cut and stained using the purified goat polyclonal antibody raised against the recombinant human Nectin-4 extracellular domain (1:60 dilution, 60 min, AF2659, R&D Systems Inc., Minneapolis, MN, USA). Whole sections of non-neoplastic breast tissues from 30 patients were also stained. As positive controls of Nectin-4 expression, skin and nipple sections were used.18 (link) Antigen retrieval was performed by microwave treatment at 750 W for 10 min in 1 M urea buffer (pH 8.0). The LSAB kit (K0679, Dako, Glostrup, Denmark) was used for signal amplification. In control sections, the specific primary antibody was replaced with isotype-matched immunoglobulins. The following antibodies were used for the identification of tumor subtypes, as previously detailed:31 (link) the anti-ER-α MoAb 6F11 (Novocastra, Menarini, Florence, Italy), the anti-PR MoAb 1A6 (Menarini), the anti-Ki67 MoAb MIB-1 (Dako) and the anti-HER-2 (Herceptest, Dako). Immunohistochemical analysis was done by two pathologists (MP, RL) by consensus without knowledge of the clinicopathological information.
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