Ecl super signal system

Duodenum segments were rinsed in ice cold Phosphate Buffered Saline (PBS) + 5 mmol/L EGTA (pH 7.42) and mucosal scrapings were prepared as described by Wang et al.^{(25 (link))} Total protein concentration was measured with a Detergent Compatible Protein Assay (BioRad Laboratories, Hercules CA) according to the manufacturer’s instructions and Western Blot analysis was conducted as described elsewhere.^{(26 )} Samples from individual mice and a pooled tissue sample for each genotype were analyzed. Briefly, 30 µg of protein were separated onto a 12.5% Tris-HCL precast Gel (BioRad Laboratories, Hercules CA) and transferred onto a 0.45 µm PVDF membrane (EMD Millipore, Billerica, MA). For the primary antibodies the membrane was incubated with anti-VDR (1:500 dilution, D-6 Santa Cruz Biotech, Dallas, TX)^{(25 (link))}, or anti-βactin (1:1000 dilution, Sigma-Aldrich, St. Louis, MO). The membrane was then incubated with the HRP-conjugated goat anti-mouse IgG light chain, secondary antibody (1:5000 dilution; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The HRP signal was detected with the ECL Super Signal system (ThermoScientific, Rockford, IL). The relative band densities were analyzed using Image J 1.48v software (NIH).