K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC Figure 2. K13 activates NF-kB pathway without involving TAK1. A. Immunoblot showing expression of ectopically expressed FLAG-tagged K13 in TAK1+/+ and TAK12/2 MEFs and endogenous K13 in BC1 cell line. B. Status of NF-kB pathway, as measured by an EMSA in TAK1+/+ and TAK12/2 MEF cells stably expressing vector and K13. The position of the induced NF-kB complexes is marked by an asterisk, while an arrow marks the position of the constitutive complexes. C. The NF-kB subunits composition of the K13-induced nuclear NF-kB complexes in the wild-type and TAK12/2 MEFs was determined with an ELISA-based DNA-binding assay performed in triplicate. Asterisks (*) indicate significance at levels of p#0.05. D. TAK1+/+ and TAK12/2 MEFs expressing FLAG-K13-ERTAM were transfected with NF-kB-Luc and Renilla reporter constructs and subsequently treated with 4OHT (20 nM) for 48 hours and the reporter assays performed as described in Figure 1A. Asterisks (*) indicate significance at levels of p#0.05. E. Nuclear extracts from Jurkat, BC1, BCBL1 and Namalwa were used for the measurement of p65 DNA-binding activity using an ELISA-based assay. F. Lack of phosphorylation of TAK1 by K13. Cell lysates prepared from BCBL1 and Namalwa cells expressing an empty vector or FLAG-K13 were probed with an antibody to detect phosphorylation of TAK1. Vector cells treated with TNFa were used a positive control. The blot was re-probed with FLAG and Tubulin antibodies to check the expression of the transduced K13 and equal protein loading, respectively. G2H. Treatment with TAK1 inhibitor 5Z-7- oxo-zeaenol had no effect on 4OHT induced NF-kB-Luc activity in 293-K13-ERTAM-NF-kB-Luc cells but effectively blocked TNFa and IL1b induced NF- kB-Luc activity. doi:10.1371/journal.pone.0036601.g002 of linear polyubiquitination in K13-induced NF-kB activity, we reconstituted NEMO-deficient MEFs with an empty vector, wild- type NEMO, NEMO-V293A/Y301A/K302A and NEMO- F305A using retroviral mediated gene transfer (Fig. 5A). In the resulting cells, we stably expressed an empty vector and K13 (Fig. 5B). After confirming the equivalent expression of the transduced proteins by Western blotting, we examined the status of the NF-kB by measuring nuclear p65/RelA DNA binding activity. Ectopic expression of K13 failed to induce nuclear p65/ RelA DNA binding activity in the NEMO2/Y MEFs reconstituted with an empty vector, but resulted in approximately 3 fold increase in the nuclear p65/RelA DNA binding activity in those reconstituted with the wild-type NEMO, thereby confirming that NEMO is required for K13-induced NF-kB activation (Fig. 5C). More importantly, we observed an equivalent increase in K13- induced nuclear p65/RelA DNA binding activity in NEMO2/Y MEFs reconstituted with NEMO-V293A/Y301A/K302A and NEMO-F305A mutants, respectively (Fig. 5C). Thus, binding of NEMO to linear polyubiquitin chain is not required for K13- induced NF-kB activation. CYLD Fails to Block K13-induced NF-kB Activity The deubiquitylating enzyme CYLD, a negative regulator of the NF-kB pathway, can cleave linear ubiquitin chains [56,57]. PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36601