TRAIL activates TAK1/NF-κB signaling in OCs. (A) Mature OCs from RAW264.7 cells and human MM cell lines, MM.1S and INA-6, were starved with 1% FBS for 24 hours and then incubated with TRAIL at 100 ng/mL for the indicated time periods. The expression of phosphorylated TAK1 (p-TAK1) and TAK1 was detected by western blotting. β-actin was used as a protein loading control. (B) Mature OCs from RAW264.7 cells were starved with 1% FBS for 24 hours and then incubated with or without the TAK1 inhibitor LLZ1640-2 (LLZ) at 300 nM, followed by the addition of TRAIL at 100 ng/mL for the indicated time periods. The expression of p-TAK1, TAK1, and phosphorylated IκBα (p-IκBα) was detected by western blotting (left). β-actin was used as a protein loading control. To observe p65 localization, the cells were fixed and stained with anti-p65 antibody (green), and their nuclei were stained with Hoechst 33342 (red). These images are taken by wide field-of-view fluorescence microscope (BX50, Olympus). Bar represents 100 μm. (C) Mature OCs from RAW264.7 cells were cultured in the presence or absence of LLZ at 300 nM for 48 hours. TRAIL was added as indicated. The protein levels of Pim-2 were analyzed by western blotting. β-actin was used as a protein loading control. (D) Mature OCs from RAW264.7 cells were cultured in the presence or absence of TRAIL (100 ng/mL) for 6 and 24 hours to perform RT-PCR and western blotting, respectively. LLZ at 300 nM and BAY11-7085 at 20 nM (upper), and the Sp1 inhibitor TMP at 100 μM (lower) were added as indicated. c-FLIP expression was analyzed. c-FLIP mRNA expression was also analyzed by real-time PCR. The results are expressed using Δ-Δ Ct method. GAPDH and β-actin served as loading controls, respectively.