K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC expressing FLAG-K13 were examined for NF-kB activation by Western blot analysis using antibodies against phospho-IkBa, Total IkBa, A20 and RelB. The blot was re-probed with FLAG and Tubulin antibodies to check the expression of the transduced K13 and equal protein loading, respectively. doi:10.1371/journal.pone.0036601.g003 As another independent test to rule out the involvement of linear Ub chains in K13-induced NF-kB activation, we tested the ability of CYLD to block K13-induced NF-kB-Luc reporter activity. We used TNFR1, CD40, and EDAR (Ectodermal Dysplasia Receptor) as controls for this experiment since NF-kB activation by these receptors is known to require linear polyubiquitination or is inhibited by CYLD [58,59]. As shown in Figure 6A-D, coexpression of CYLD had no effect on K13-induced NF-kB activity but effectively blocked TNFR1, CD40 and EDAR- induced NF-kB activity. Thus, in contrast to the receptors of the TNFR family, K13-induced NF-kB is not dependent on linear ubiquitin chains. Essential Role of NEMO in Mediating K13-IKK Interaction and IKK Activation We and others have previously demonstrated that K13 interacts with NEMO and NEMO is essential for K13-induced NF-kB activation [34,60]. The studies in the preceding section clearly demonstrate that TAK1, TRAF6 and linear polyubiquitination of NEMO are not involved in K13-induced NF-kB activation. Therefore, to explain the role of NEMO in K13-inducd NF-kB activation, we tested the hypothesis that NEMO mediates the interaction between K13 and IKK1 and IKK2. For this purpose, FLAG-tagged K13 was immunoprecipitated from the WT and NEMO-deficient Jurkat cells using Flag or control antibody beads and its interaction with endogenously expressed NEMO, IKK1 and IKK2 was examined by Western blot analysis. As shown in Figure 4. SHARPIN is not required for K13-induced NF-kB activation. A. MEFs deficient in SHARPIN (cpdm) were able to activate NF-kB reporter activity in the presence of K13, but not with TNFa. Asterisks (*) indicate significance at levels of p#0.05. B. SHARPIN is not required for K13- induced NF-kB p65 DNA binding as determined by ELISA. Asterisks (*) indicate significance at levels of p#0.05 as compared to vehicle-treated cells. C. Total cell lysates from wild-type, and cpdm MEF stably expressing an empty vector or FLAG-tagged K13 were used to examine the requirement of SHARPIN on K13-induced NF-kB activation by upregulation of expression of A20, RelB, phosphorylation of IkBa and degradation of total IkBa. The expression of the FLAG-tagged K13 protein and equal protein loading was confirmed by blotting with FLAG and Tubulin antibodies, respectively. doi:10.1371/journal.pone.0036601.g004 PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36601