Anti vdr antibody

A SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003, Cell Signaling Technology) was used according to the manufacturer’s instructions. For each immunoprecipitation, a total of 4 × 10⁶ SW480 cells and 1.22 μg of anti-VDR antibody (#12550, Cell Signaling Technology) were used, and the same amount of normal rabbit IgG was used as the negative control and 10 μl histone H3 rabbit antibody was used as the positive control. RT-qPCR was used to verify the binding sites between VDR and the promoter of NAT2. The data were calculated as relative enrichment against the negative control. The primers used are listed in Supplementary Table S1.

Cells were washed twice with phosphate-buffered saline (PBS) and lysed with Triton X-100 lysis buffer (20 mM TRIS–HCl pH 7.0, 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, 1% Triton X-100, 2 mM orthovanadate, 10 nM okadaic acid, protein inhibitor mix (2 ng/ml of antipain, aprotinin, chymostatin, leupeptin, pepstatin and trypsin inhibitor, each), 40 μg/ml phenylmethylsulfonyl fluoride). Cells were centrifuged for 10 min at full speed at 4 °C. The supernatant was transferred and after determination of protein concentration by the Bradford assay, equal amounts of protein were boiled in sample buffer and separated by SDS-PAGE (8%). The gels were blotted onto a nitrocellulose membrane and blocked in Rotiblock (Carl Roth). The anti-VDR antibody (Cell Signaling Technology, #12,550) and the β-actin antibody (Cell Signaling Technology, #4970S) were used in a 1:1000 dilution. The blots were developed with infrared-fluorescent-dye-conjugated secondary antibodies (LI-COR Biosciences) and detected with an Odyssey Classic system (LI-COR Biosciences).

Immunohistochemistry for VDR was performed in fixed and paraffin-embedded sections (5 µM) of intestinal resections from damaged mucosa of CD patients or healthy mucosa from colorectal cancer patients [23 (link)].
The heat-mediated antigen retrieval was performed with 10 mM of sodium citrate buffer at pH 6.0 (Dako Target Retrieval Solution) during 20 min at 98 °C. After the inactivation of endogenous peroxidase and blocking the slides during 1 h at room temperature, intestinal tissues were incubated with the primary antibody Anti-VDR antibody (1:200; 12550, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. The samples were incubated for 30 min at room temperature with the Biotinylated Universal Antibody (1:100; BA-1400, Vector, Peterborough, UK) as a secondary antibody. A negative control without the primary antibody incubation was also performed. Afterwards, in order to get a stronger signal, a 30 min room temperature incubation with Vectastain^® Universal Elite ABC Kit (Vector) was performed. A signal was developed after 2 min with DAB enhanced substrate (Sigma-Aldrich, Madrid, Spain). All samples were counterstained with hematoxylin, and pictures were obtained with a microscope (Leica DMI 3000).