TRAF2 degradation is required for macrophage differentiation. (A) (Top panel) Immunoblot analysis of c-IAP1 expression in human monocytes transfected 48 hours before with control (Co) or cIAP1-specific (1 and 2) siRNAs. (Bottom panel) Flow cytometry analysis of CD71 expression at the surface of human monocytes transfected with control (gray area) or cIAP1 targeting siRNA sequences 1 (black line) and 2 (dark gray line) and exposed 48 hours later to 100 ng/mL M-CSF for 48 hours. (B-E) THP-1 cells were transduced with an empty retroviral vector (Co) or a vector encoding a proteasome-resistant TRAF2 mutant (TRAF2*) or a vector encoding a cIAP1ΔRING mutant (ΔRING), then induced to differentiate into macrophages by exposure to 20 nM TPA for the indicated times. (B) Immunoblot analysis of TRAF2 expression in transduced cells exposed for the indicated times to 20 nM TPA. HSC70 indicates loading control. (Bottom panel) TRAF2/HSC70 ratio of signal intensities quantified by densitometry (○ indicates control vector; and ●, TRAF2*). (C) Transduced cells were left untreated (top panel) or exposed to 20 nM TPA for 48 hours (bottom panel) before flow cytometry analysis of CD11b membrane expression. Gray area represents control vector; black lines, TRAF2*-transfected cells. (D) Engulfment activity of labeled bacteria E coli was measured in transduced cells exposed for 48 hours to 20 nM TPA. Results were normalized to control cells (mean ± SD of 3 independent experiments). *Statistically significant differences (P < .05, Student test). (E) NF-kB DNA-binding activity was assessed by EMSA in THP-1-transduced cells exposed for the indicated times (hours) to 20 nM TPA. A representative experiment is shown.