(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).