CARP2 deficiency does not alter induction of NF-κB by TNFα Afsar U. Ahmed, Maryline Moulin, Franck Coumailleau, W. Wei-Lynn Wong, Maria Miasari, Holly Carter, John Silke, Michel Cohen- Tannoudji, James E. Vince, David L. Vaux  Current Biology  Volume 19, Issue 1, Pages R15-R17 (January 2009) DOI: 10.1016/j.cub.2008.11.040 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 Requirement for CARP2 for TNFα-induced NF-κB. (A) Loss of CARP2 does not influence TNFα-induced phosphorylation of p65. Wild-type (WT) and Carp2/Rififylin-deficient MEFs were incubated with 100 ng/ml of Fc–TNFα for the indicated times and analysed for phosphorylation of p65 (P-p65) by western blotting. Similar results were observed with a lower (10 ng/ml) dose of Fc–TNFα (Figure S2D). (B) Carp2/Rififylin-deficient cells show normal IκBα degradation and NF-κB-dependent IκB recovery in response to TNFα. WT or Carp2/Rififylin-deficient MEFs were treated with Fc–TNFα (100 ng/ml) for the indicated times and IκBα levels examined by western blotting. Similar results were observed with a lower (10 ng/ml) dose of Fc–TNFα (Figure S2D). (C) Inducible CARP2 overexpression does not influence IκBα degradation or NF-κB-dependent IκB recovery in response to TNFα. Stable FLAG–CARP2 inducible D645 cells were induced for 24 h and then treated with Fc–TNFα (100 ng/ml) for the indicated times and IκBα levels were examined in the absence or presence of FLAG–CARP2. (D) Endogenous RIP1 protein levels are normal in Carp2/Rififylin-deficient cells. Five independent WT (C2+/+) or Carp2/Rififylin-deficient (C2−/−) primary MEF lines were treated with or without Fc–TNFα (100 ng/ml) for 30 min and endogenous RIP1 levels analysed by western blotting. (E) TNFR1-associated RIP1 modification is similar in WT and Carp2/Rififylin-deficient MEFs. Fc–TNFα was used to immunoprecipitate TNFR1 and associated RIP1 at the indicated times following Fc–TNFα treatment. The immunoprecipitated TNFR1 complex was analysed by western blotting with the indicated antibodies. Time 0 represents cells where Fc–TNFα was added after cells were lysed. The majority of TNFR1 resides intracellularly and only a small portion is accessible to Fc–TNFα when applied to intact, live MEFs: compare anti-TNF and anti-TNFR1 blots at time 0 with time 15 and 60 min. The experiment was repeated on independent WT and Carp2/Rififylin-deficient MEF lines with the same result (Figure S2G). Representative western blots of the cell lysates used for these immunoprecipitation experiments are shown in Figure S2H. RIP1U: unmodified RIP1. RIP1M: modified (ubiquitylated) RIP1. (F,G) TNFα induction of NF-κB reporter activity is normal in Carp2/Rififylin-deficient and CARP2-overexpressing cell lines. (F) WT and Carp2/Rififylin-deficient MEFs (left graph) or stable FLAG–CARP2-inducible D645 cells (right graph) were infected with a lentiviral destabilised-luciferase NF-κB reporter. Cells were treated with Fc–TNFα (100 ng/ml) and reporter activity measured 24 h later and depicted as fold change in reporter activity relative to untreated cells. Error bars represent SD of at least three independent clones. (G) Cells were treated in the same manner as in (F), except they were infected with an NF-κB–GFP reporter plasmid and NF-κB activation assessed by flow cytometry. (H) NF-κB target gene regulation in WT and Carp2/Rififylin-deficient cells. WT and two independent Carp2/Rififylin-deficient MEF cell lines were treated with Fc–TNFα (100 ng/ml) for 24 h and the RNA extracted and RT-PCR performed for the indicated transcripts. Current Biology 2009 19, R15-R17DOI: (10.1016/j.cub.2008.11.040) Copyright © 2009 Elsevier Ltd Terms and Conditions