Bcl 3

Cells were placed on ice and washed once with cold PBS before being harvested in cold 1 × lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton-x-100, complemented with 40 μl/ml complete protease inhibitors (Roche Applied Science). Lysates were cleared by centrifugation at 12,000 × g for 10 min at 4 °C and protein content was determined using a Bradford assay according to manufacturer’s instructions (Thermo Fisher Scientific). Equal amounts of protein were electrophoretically separated on 10% SDS/polyacrylamide gels and proteins were transferred onto Immobilion-FL polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% non-fat milk in PBS-T for 1 h at room temperature followed by overnight incubation at 4 °C with primary antibodies against α-tubulin (1:4000, Sigma), Bcl-3 (1:500, Santa Cruz), cyclin D1 (1:1000, Abcam). Primary antibodies were detected with horseradish peroxidase-labeled secondary antibodies (1:5000, Dako). Chemiluminescence was detected with a charge-coupled device camera (Fujifilm).

Cells were placed on ice and the media were aspirated. The cells were washed once with cold phosphate-buffered saline (PBS) and harvested in cold 1 × lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton-100, containing 40 μl/mL complete protease inhibitors (Roche Applied Science)]. Lysates were cleared by centrifugation at 12 000 × g for 10 min at 4°C and the protein content was determined. Equal amounts of protein were electrophoretically separated on 10% SDS/polyacrylamide gels and proteins were transferred onto Immobilion-FL PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk in PBST for 1 hour at room temperature followed by overnight incubation at 4°C with primary antibodies against Actin (1:40000 MP Biomedicals), α-tubulin (1:4000 Abcam), Lamin B (1:1000 Santa Cruz), Bcl-3 (1:500 Santa Cruz). Primary antibodies were detected with horseradish peroxidase-labelled secondary antibody (1:5000, DAKO). The chemoluminescence was detected with a charge-coupled device camera (Fujifilm).

Frozen or paraffin tissue sections were processed for Immunofluorescent staining. Non-specific binding sites were blocked by 2.5% goat serum in bovine serum albumin (0.5% w/v) for 1 hour. The sections were then incubated with the following antibodies for overnight at 4°C: GR-1 (BD Biosciences, 1:100); CD11b (BD Biosciences, 1:100); CD11b (Abcam, 1:100); CD16 (Abcam, 1:100); CD11c (Abcam, 1:100); CD4 (R&D system, 1:50); CD4 (BD Biosciences, 1:100); MHCII (Abcam, 1:100); FOXP3 (Abcam, 1:100); Vitamin D receptor (VDR; Abcam, 1:100) Bcl-3 (Santa Cruz, sc-13038, 1:100); NFκB-p50 (Santa Cruz, sc-114, 1:100); NFκB-p52 (Santa Cruz, sc-298, 1:100); NFκB-p65 (Santa Cruz, sc-109, 1:100); Gli1 (Santa Cruz, sc-20687, 1:100); Cyclin D1 (Neomarkers, RM-9104-s1, 1:100); N-cadherin (Santa Cruz, sc-7939, 1:100) and K17 (Neomarkers, MS-489-S1, 1:100). After washing 3 times with PBS, the sections were incubated with Alexa Fluor^® 488 or Alexa Fluor^® 594 secondary antibodies (Life Technologies) for 1 hour at room temperature. After removal of antibodies, the sections were rinsed with PBS and mounted with mounting medium containing DAPI. Fluorescence was immediately recorded on an Olympus EX51 microscope.