Control igg

Fresh whole cells lysate in Tween-20 Co-IP buffer were immunoprecipitated with anti-NEDD4L (Abcam, Ab240753), anti-c-Myc (Abcam, Ab32072) or control IgG (Santa Cruz Biotech., Santa Cruz, CA, USA) antibodies and protein A/G-agarose beads over night at 4°C. Precipitates were rinsed three times with ice-cold Co-IP buffer and subjected to western blot to determine the pull-down efficacy.

To analyse RUNX2 promoter activity, Nthy-Ori 3-1, TPC1, and BHP10-3 cells were plated on 24-well plates at a density of 2 × 10⁴ cells/well 1 day before transfection. Plasmid DNA was mixed with TransIT-2020 (Mirus, Madison, WI) and transfected into the cells following the manufacturer’s protocol. After 48 h, cells were washed twice with 1× PBS and then lysed in reporter lysis buffer (Promega, Madison, WI). Cell extracts were subjected to assays using a luciferase assay system (Promega, Madison, WI) according to the manufacturer’s instructions. Luciferase activity was measured in triplicate, averaged, and then normalized to β-galactosidase activity using o-nitrophenyl-β-D-galactopyranoside (Sigma-Aldrich) as a substrate.
ChIP assays were performed with a ChIP kit (Upstate Biotechnology, Lake Placid. NY), with modified instructions from the manufacturer, using antibodies against HOXA9 (Proteintech, Rosemont, USA) or control IgG (Santa Cruz Biotechnology, Santa Cruz, CA). The precipitated DNA was subjected to PCR amplification with specific primers for the RUNX2 P1 promoter region, which contains HOXA9-binding sites. The following primers were used for PCR: RUNX2 P1 sense, 5′-GCAAAAAGGCAGAGGTTGAG-3′; RUNX2 P1 antisense, 5′-CCCCCTTGCTCTTTCTCTCT-3′.

Mouse proximal jejunum (10 cm) was collected, washed in PBS, and cut in 2–3 mm pieces in
chelating buffer (PBS, 1 mm-DTT (dithiothreitol), 5 mm-EDTA). Jejunal
fragments were thoroughly vortexed before and after two incubations (20 min at 37 °C under
agitation) in the chelating buffer. Epithelial cell suspension was then filtered on a 70
μm cell strainer on top of complete medium (Roswell Park Memorial Institute (RPMI)-1640
medium, fetal calf serum (FCS) 10 %, penicillin-streptomycin (PS) 1 %). After
centrifugation, cells were re-suspended in complete medium. After fixing in 4 %
paraformaldehyde (PFA), 10⁶ cells were incubated 30 min with the primary GLP-1
antibody (no. sc-7782; Santa Cruz) or control IgG (no. sc-2028; Santa Cruz) and a
Cy5-coupled secondary antibody (Jackson Immunoresearch) diluted in a permeabilisation
buffer (PBS, FCS 2 %, saponin 0·1 %). Stained cells in suspension were analysed with a BD
LSRII cytometer and expressed as a percentage of total epithelial cells.