cIAP1 is responsible for differentiation-associated TRAF2 depletion. (A) Coimmunoprecipitation of cIAP1 and TRAF2 in U937 cells exposed for the indicated times to 20 nM TPA. (Right panel) MG132 was added during the last 7 hours of exposure to TPA (IP with an anti-cIAP1 antibody; immunoblot with anti-TRAF2 and anti-cIAP1 antibodies; Ig indicates irrelevant immunoglobulin). (B) U937 cells were left untreated or treated with 20 nM TPA for 48 hours in the absence or presence of 50 μM Smac-N7 peptide before immunoblot analysis of TRAF2. (C) U937 cells were transduced with murine stem cell virus–based retroviral vector, either empty (Control) or encoding cIAP1 or ΔRING (a cIAP1 construct deleted of the C-terminal E3-ubiquitin ligase domain), and cIAP1 expression was checked by immunoblotting (top panel) before inducing cell differentiation by exposure to 20 nM TPA for the indicated times and analyzing TRAF2 expression by immunoblot (bottom panel). (D) THP-1 cells were transfected with either negative-control (Co) or 2 cIAP1 targeting siRNA sequences (1 and 2), 48 hours before exposing the cells to 20 nM TPA. (Left panel) Immunoblot analysis of cIAP1, 48 hours after siRNA treatment. (Right panel) Immunoblot analysis of TRAF2 at indicated times after TPA exposure (20 nM). (E) Macrophages were obtained by exposure of human blood monocytes for 6 days to M-CSF (100 ng/mL) and then transfected with either negative-control (Co) or cIAP1 targeting siRNAs (1 and 2). cIAP1 (left panel) and TRAF2 (right panel) expression was studied by immunoblotting, 48 hours after siRNA transfection in independent samples (A-C). (B-E) HSC70 used as loading control.