TRAF2 is required for macrophage differentiation. (A,B,D,E) THP-1 cells or human monocytes were transfected with either negative-control (Co) or TRAF2 targeting siRNAs (sequences 1 and 2), 48 hours before exposing the cells to differentiating agents (20 nM TPA for THP-1 and 100 ng/mL M-CSF for human monocytes). (A) (Top panel) Immunoblot analysis of TRAF2, 48 hours after siRNA treatment. (Bottom panel) Flow cytometry analysis of CD11b or CD71 membrane expression, 48 hours after addition of differentiation inducers. Gray histogram represents negative control siRNA; white histograms, TRAF2-targeting siRNA sequence 1 (black line) and 2 (gray line). One representative of 3 experiments is shown. (B) Fluorescence microscopy analysis of NF-κB p65 subunit (green) in THP-1 cells exposed for the indicated times to 20 nM TPA (original magnification ×400). Nuclei were stained using Hoechst (blue). (C) Immunoblot analysis of Iκ-Bα in THP-1 cells exposed for the indicated times to 20 nM TPA. (D) Immunoblot analysis of Iκ-Bα in THP-1 cells exposed for the indicated times to 20 nM TPA, 48 hours after transfection with indicated siRNAs. (E) Percentage of cells with nuclear p65 quantified by immunofluorescence in THP-1 cells exposed for the indicated times to 20 nM TPA, 48 hours after treatment with scramble (black) or TRAF2-specific (gray represents sequence 1; white, sequence 2) siRNAs. Data are mean plus or minus SD of 3 independent experiments.