Anti β catenin 610154

A total of 2 × 10⁷ cells per cell line were used for the chromatin immunoprecipitation (IP), which was performed as described previously (Kumar et al., 2014 ). The 1% formaldehyde cross-linked chromatin was isolated and sheared into 500–1000 bp fragments by sonication (Bioruptor UCD-200, Diagenode; New York, NY, USA). Prior to IP, 1% of the sonicated DNA was taken as input. The concentrations of antibodies used for overnight incubation at 4°C were as follows: 2.5 µg of anti-β-catenin #610154 (BD Biosciences), -, 2 µg of IgG (negative control). Primers for TCF/LEF included Site #1, #2, and #3 on the MUC4 promoter; the TCF/LEF site on the c-myc promoter (positive control) and primers for the promoter of an unrelated gene (negative control) were also used (Supplementary Table B.2.). Immunoprecipitated qPCR Ct (cycle threshold) values were normalized to input Ct values, and all data are represented as a percentage of input.

The UNMC Rapid Autopsy Program was used to obtain tissue microarrays (TMA) containing primary PDAC tissue specimens (IRB-091-01) and normal pancreas, kidney, and colon tissue spots as controls (n = 30, 25 PC spots). TMAs containing metastatic spots (n = 26; 25 liver metastatic spots, 1 lung metastatic spot) with normal pancreas, colon, and kidney tissues as controls were also used (IRB-091-01). Prior consent was obtained from all participants in the Rapid Autopsy Program, and the UNMC Institutional Review Board (IRB) approved this study. The following antibodies were used: anti-β-catenin #610154, (BD Biosciences; San Jose, CA, USA) anti-MUC4 monoclonal antibody clone 8G7 (developed in our lab). The protocols used for immunohistochemical (IHC) detection of β-catenin and MUC4 have been described previously (Barker and van den Born, 2008 (link); Kaur et al., 2014 (link)). The tissue microarrays (TMAs) were evaluated by a UNMC pathologist and were given a composite score ranging from 0 – 12, which was a product of the intensity of staining (range 1 – 3) and number of cells stained (range 1 – 4; 0 – 25% area stained = score of 1, 26 – 50% = score 2, 51 – 75% = score 3, and 76 – 100% = score 4).