(A) and (B). CARP-2 localizes to endocytic membrane vesicles. Confocal images of HeLa cells immunostained with control or CARP-2 (Red) (A), or CARP-2 (Red) and Rab 5 (Green) antibodies (B). Scale bar is 10 μm. Areas of colocalization was shown by line intensity profile.
(C). CARP-2 is recruited to TNF-R1 receptosomes. Magnetic TNF-R1 fractions and post-nuclear extracts (lysate) from HeLa cells were probed for indicated proteins.
(D). Internalized TNF-R1 vesicles associate with CARP-2 positive vesicles. Confocal images of HeLa cells labeled with Biotin-TNF/FITC (Green) conjugated streptavidine and anti-CARP-2 (Red). Colocalization analyses were performed with Zeiss AIM software v3.2 to give the overlay image of colocalized pixels, scatter plots and Pearson's coefficients. Ch 3 is FITC (green) and Ch4 is CY3 (Red). Scale bar is10 μm.

(A) and (B). Inhibitors of internalization and MG-132 but not Z-VAD FMK rescue TNF–induced RIP degradation. Expression of endogenous RIP in TNF-stimulated NEMO −/− MEFs, pretreated with medium or FILIPIN (2 μg/ml) or chlorpromazine (CPZ) (1.6 μg/ml, 3.2 μg/ml, 6.4 μg/ml) (A), or with indicated compounds (B) is shown.
(C). CPZ inhibits TNF–induced RIP ubiquitination. RIP from extracts of TNF-stimulated NEMO −/− MEFs pretreated with MG-132 and medium (UT) or CPZ (10 μg/ml) was immunoprecipitated under denaturing conditions and probed for ubiquitin and RIP.
(D). Knockdown of CARP-2 rescues TNF-induced RIP loss. Extracts of TNF-stimulated NEMO−/− MEFs expressing ShRNA constructs were probed for indicated proteins.
(E). Ubiquitinated RIP accumulates on TNF-R1 endocytic vesicles in the presence of MG-132. Magnetic TNF-R1 fractions from U937 cells were probed for indicated proteins.
(F). CARP-2 targets TNF receptor-associated RIP for degradation. Extracts from TNF-stimulated HeLa cells expressing ShRNA constructs were precipitated with hamster anti TNF-R1 antibody and probed for indicated proteins.

(A). CARP-2 binds RIP kinase domain. Extracts from 293T cells expressing FLAG tagged RIP kinase domain (KD) or without kinase domain (ΔKD) were incubated with extracts from cells expressing vector or wild type V5-CARP-2. RIP was immuno-precipitated and probed for CARP-2.
(B). Kinase domain is essential for CARP-2 mediated RIP loss. Extracts from HeLa cells transfected with pcDNA3-nV5-RIP Full length (FL) or ΔKD together with CARP-2 were probed for indicated proteins.
(C). Kinase domain is essential for CARP-2 mediated RIP degradation. 293T cells transfected with pcDNA3-nV5 -RIPΔKD and pFLAG-CMV-CARP2 variants were analyzed by procedure described in figure 5C.
(D). RIP kinase domain negatively regulates TNF-induced NF-κB activation. Extracts of medium (Med) or TNF treated RIP−/− MEFs transfected with indicated constructs in pMSCV vector along with reporter plasmids were assayed for the reporter activities.

(A). Schematic diagram shows the domain structure and position of conserved Histidine 333 in the RING domain of CARP-2.
(B) and (C). CARP-2 inhibits TNF-induced IKK activation and IκB α degradation. Extracts of 293T cells transfected with indicated plasmids and treated with TNF for 5 min (B), or as indicated (C) were subjected to in vitro IKK kinase assay (B), or probed for expression of indicated proteins (B and C).
(D) and (F). CARP-2 inhibits TNF-induced NF-κB reporter activity. Extracts of TNF-stimulated 293T cells expressing reporter plasmids along with different amounts of FLAG-CARP-2 variants (D), or ShRNA constructs (F) were assayed for reporter activities and probed for CARP-2.
(E). CARP-2 inhibits TNF-induced IL-6 production. IL-6 secreted from mouse embryonic fibroblasts (MEFs) expressing CARP-2 variants was measured by ELISA and expression of CARP-2 and β-actin in the extracts is shown.
(G) and (H). Knockdown of CARP-2 prolongs TNF-induced IKK activation and IκB αdegradation. Extracts of TNF-treated 293T cells expressing ShRNA constructs were subjected to in vitro IKK kinase assay (G), or probed for expression of indicated proteins (G and H).

(A). CARP-2 inhibits NF-κB activation induced by early TNF-R1 complex components. Extracts from 293T cells transfected with indicated constructs along with reporter plasmids were assayed for reporter activities. The fold increase in the activity relative to vector background is shown in parenthesis. Expression of signaling molecules and one representative set of CARP-2 are shown.
(B) and (C). CARP-2 downregulates, and lactacystin rescues RIP expression. Expression of exogenous proteins in the extracts of 293T cells transfected with indicated constructs and treated with proteasomal inhibitor lactacystin are shown.
(D). CARP-2 binds to endogenous RIP. Extracts from 293T cells expressing FLAG-CARP-2 variants were immunoprecipitated with anti-FLAG antibody and probed for indicated proteins.
(E). Endogenous CARP-2 binds and promotes endogenous RIP degradation in TNF-stimulated cells. CARP-2 from extracts of TNF-stimulated HeLa cells was immunoprecipitated and probed for RIP and CARP-2.
(F). Endogenous CARP-2 binds endogenous RIP in TNF receptosomes. TNF receptosomes isolated from MG-132 treated HeLa cells were subjected to immunoprecipitation with anti-CARP-2 antibody coupled to magnetic MACS microbeads or with anti-rabbit IgG magnetic microbeads. RIP and CARP-2 in total receptosomes are shown.

(A). CARP-2 promotes TNF-induced endogenous RIP ubiquitination. Purified Hisubiquitinated proteins from TNF-stimulated 293T cells transfected with indicated plasmids and treated with MG-132 were analyzed with anti-RIP antibody (left panel). 293T cells expressing pcDNA3-HA-ubiquitin and pCMV-tag2B-RIP-His along with indicated plasmids were pretreated with MG-132. His-RIP was purified and analyzed for ubiquitination (right panel).
(B). CARP-2 mediates K48-linked polyubiquitination of endogenous RIP. Endogenous RIP from TNF-stimulated 293T cells expressing indicated plasmids, preincubated with MG-132, was immunoprecipitated under denaturing conditions and probed for ubiquitin and RIP.
(C). CARP-2 promotes RIP degradation. 293T cells transfected with pFLAG-CMV-RIP and pcDNA3-nV5-CARP2 variants were incubated with DMEM containing [³⁵S] ProMix for 30 minutes and chased by growing in complete serum medium. RIP from the extracts was immunoprecipitated and analyzed by autoradiography and PhosphorImager.
(D). In vitro ubiquitination of RIP by CARP-2. Modified RIP band is indicated.

Model for regulation of TNF mediated NF-κB activation by CARP-2