TRAF2 is degraded in the cytoplasm. (A) Immunoblot analysis of cIAP1 in nucleus and cytoplasm-enriched fractions and TRAF2 in whole-cell extracts from THP-1 cells undergoing differentiation on TPA exposure (20 nM for the indicated times). HSC70 indicates loading control. (B) THP-1 cells were induced to differentiate into macrophages by exposure to 20 nM TPA for 24 hours, in the absence or presence of 100 nM leptomycin B (LMB) before analyzing cIAP1 localization by fluorescence microscopy (top panel, c-IAP1 in green). Nuclei were stained using Hoechst blue. (Bottom panel) Immunoblot analysis of TRAF2 expression. HSC70 indicates loading control. (C) Immunoblot analysis of TRAF2 in nucleus and cytoplasm-enriched fractions from TPA-treated THP-1 cells (20 nM for the indicated times). Poly(ADP-ribose) polymerase was used as a nucleus fraction marker and X-linked inhibitor of apoptosis protein as a cytoplasm fraction marker. HSC70 indicates loading control. (D) Fluorescence microscopic analysis of TRAF2 expression (green) in THP-1 cells exposed for the indicated times to 20 nM TPA (original magnification ×400). (E) Confocal fluorescence microscopic analysis of TRAF2 (red) and cIAP1 (green) or TRAF2 (red) and GM130 (green) expression in undifferentiated (Control) and TPA-differentiated (24 hours) THP-1 cells, in the absence or presence of 30 μM MG132 or 10 μM lactacystin added for the 7 or 4 last hours of treatment, respectively. Nuclei were stained using Hoechst blue (original magnification ×500).