Response: CARP1 regulates induction of NF-κB by TNFα

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Presentation transcript:

Response: CARP1 regulates induction of NF-κB by TNFα Wentao Liao, Ken-ichi Fujita, Qi Xiao, Vladimir Tchikov, Wensheng Yang, Michele Gunsor, Susan Garfield, Paul Goldsmith, Wafik S. El- Deiry, Stefan Schutze, Srinivasa M. Srinivasula Current Biology Volume 19, Issue 1, Pages R17-R19 (January 2009) DOI: 10.1016/j.cub.2008.11.041 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 Role of CARP1 in TNF signaling. (A) Domain structures of CARP1 and CARP2. (B) Tissue expression of CARP1 and CARP2. Normal human tissue extracts (Prosci, Poway, CA) were probed with antibodies to detect the expression of CARP1 or CARP2 proteins and Actin. LN, lymph node; S. muscle, smooth muscle. (C) CARP1 associates with TNFR1 receptosomes. Magnetic TNFR1 fractions were immunoblotted with anti-CARP1 antibody. Post-nuclear extracts were used as controls (lysate). (D) CARP1 interacts with RIP1. CARP1 protein was immnoprecipitated from extracts of TNF-stimulated cells stably expressing CARP1–FLAG with anti-FLAG antibody and the precipitates were probed for endogenous RIP1 and CARP1. (E) CARP1 promotes ubiquitination of RIP1 with K48-linked chains. 293T cells expressing HA–ubiquitin variants along with indicated plasmids were pretreated with proteasome inhibitor MG-132 and stimulated with TNFα. RIP1 was purified from cell extracts under denaturing conditions and analyzed for ubiquitination with anti-HA antibody. The extracts were analyzed with indicated antibodies. (F) CARP1 targets receptor-associated RIP1. Extracts of 293T cells transfected with CARP1-specific or non-specific (NS) shRNA and stimulated with TNFα were immunoprecipitated with anti-TNFR1 antibody and the precipitates were probed for RIP1 and TNFR1. Expression levels in the lysates of CARP1 protein, RIP1 and β-actin are shown. (G) CARP1 expression inhibits IκBα degradation. 293T cells transfected with indicated plasmid DNAs for 24 h were treated with TNFα for 15 or 30 min and the cellular extracts were probed using anti-IκBα, anti-β-actin and anti-FLAG (for CARP1) antibodies. (H) CARP1 expression inhibits IL-6 secretion. MEFs (pools) expressing vector, CARP1 or CARP1-H342A (a RING mutant) were treated with medium (white bars) or TNF (10 ng/ml; black bars) overnight, and the supernatants were assayed for IL-6 by ELISA. Data represent the average of three experiments. The expression of CARP1 and β-actin in the cellular extracts are shown. (I) CARP1 knockdown enhances NF-κB activation. 293T cells expressing reporter gene along with control or CARP1-specific shRNA constructs for 48 h were treated with medium (UT) or TNF for 5 h, and the extracts were assayed for reporter and β-galactosidase activities. The data represent the average of at least three experiments. Current Biology 2009 19, R17-R19DOI: (10.1016/j.cub.2008.11.041) Copyright © 2009 Elsevier Ltd Terms and Conditions