Ab49470

BCL-3 CoIPs were performed in SW1463 nuclear-enriched lysates as previously described (Petherick et al., 2013 (link)). Briefly, 500 µg of rabbit pan-IgG (12-370; EMD Millipore)-pre-cleared nuclear-enriched protein lysates were cleared with 6 µg of anti-BCL-3 antibody (23959; Proteintech) conjugated to Dynabeads Protein A beads (Invitrogen) before undergoing western blot analysis. Beads were retrieved using a Dyna-Mag 2 magnet (Invitrogen). Immunoprecipitates were analysed by western blot for BCL-3 (ab49470; Abcam) and β-catenin (610153; BD Biosciences). For TNF-α-treated CoIPs, 100 ng/ml TNF-α (Source BioScience, Nottingham, UK) was added to cells for 6 h prior to lysis. Immunoprecipitates were additionally analysed for p52 (05-361; EMD Millipore).

Cell lysates were prepared and subjected to western analysis as described previously (Williams et al., 1993 (link)) using antibodies to the following: α-tubulin (T9026; Sigma-Aldrich; 1:10,000), ASCL2 (4418; EMD Millipore, Watford, UK; 1:500), β-catenin (9587; Cell Signaling Technology, MA, USA; 1:5000), β-catenin (610153; BD Biosciences, CA, USA; 1:10,000), de-phosphorylated (actively signalling) β-catenin (05-665; EMD Millipore; 1:1000), BCL-3 (ab49470; Abcam, Cambridge, UK; 1:1000), BCL-3 (23959; Proteintech, Manchester, UK; 1:2000), MYC (sc-40; Santa Cruz Biotechnology, TX, USA; 1:500), cyclin D1 (2978; Cell Signaling Technology; 1:1000), lamin A/C (4200236; Sigma; 1:5000), LEF1 (2230; Cell Signaling Technology; 1:1000), LGR5 (ab75850; Abcam; 1:1000), NF-κB1 (p50; sc-8414; Santa Cruz Biotechnology; 1:1000), NF-κB2 (p52;05-361; EMD Millipore; 1:1000) and TCF4 (2569; Cell Signalling Technology; 1:1000).

Immunohistochemistry was performed on paraffin sections of 38 ESCC cases, including eight cases with non-tumor surrounding tissue, and 22 healthy controls. For antigen retrieval, sections were incubated in a pressure cooker while submerged in citrate buffer, pH 6.0 for BCL3 staining and EDTA buffer, pH 8.0 for IL6 detection. Sections with 3 µm were then incubated in 3% hydrogen peroxide for 20 min and Protein Block solution for 30 min (Dako^®, Denmark) before the incubation with the primary antibody against BCL3 (Abcam^® – ab49470) or IL6(Abcam^® – ab6672), overnight at 4°C. Detection and staining were performed with the Novolink™ Polymer Detection System (Leica Biosystems, UK). Sections were counterstained with Harris’ hematoxylin. FFPE healthy tonsil was used as a positive control of BCL3 expression and lymph node as a positive control to IL6 detection. In the negative control, the primary antibody was replaced with the antibody diluent solution.