Nbp1 76864

HT-29 cells were propagated in RPMI-1640 with 10% FCS. HT-29 cells were exposed to L-mimosine (0-500 μmol/L) for 5 hours at 37°C. Cells were then washed with PBS. For gene expression experiments, total RNA was extracted with Trizol reagent (Life Technologies) and qPCR for selected genes was carried out as noted above. For protein analysis, protein was extracted with RIPA buffer, and concentrations determined using BCA assay (Pierce). Proteins were separated by 4-12% Tris-tricine (Invitrogen) gel electrophoresis. Western blot analysis was performed using anti HIF-1α (NB100-449, Novus) and anti LL-37 (NBP1-76864, Novus) rabbit polyconal antibodies. The secondary Ab was a horseradish (HRP) peroxidase-conjugated goat anti-rabbit (7074, Cell Signaling). Actin (pan-Actin rabbit monoclonal antibody, D18C11, Cell Signaling) was used as an internal control to confirm equal protein loading. Immunoreactive proteins were detected using the ECL-chemiluminescent system (EMD Millipore Immobilon).