Hsp60 for IKK Regulation Figure 1. Identification of Hsp60 in IKK complex. A. A silver-stained polyacrylamide gel resolving the affinity-purified IKK complex. B. MS/MS spectra of [M+2H]2+ ions of the peptides derived from the protein band corresponding to Hsp60. C. Immunoblot (IB) analyses of IKK subunits and Hsp60 in the affinity-purified complex. D. Interaction of Hsp60 with IKK complex. IKK complex was immunoprecipitated (IP) from HeLa cell lysates (500 mg total proteins for each) with IKKa, IKKb, and IKKc-specific antibodies. The IKKa/b/c subunits, Hsp60, and Hsp90 were immunoblotted. WCL, whole cell lysate. E. TNF-a-independent interaction of Hsp60 and IKK complex. F. Co-immunoprecipitation of Hsp60 and IKK complex in cytosolic fraction. Upper panel, post-nuclear supernatant (PNS), cytosol (Cyto), and mitochondria (Mito) fractions from HeLa cells were immunoblotted. COX4 and tubulin were used as mitochondrial and cytosolic markers, respectively. Lower panel, Hsp60 was immunoprecipitated from the cytosolic fraction using either control goat IgG or anti-Hsp60 antibodies (K-19 and N-20). Representative blots are shown (n = 3). doi:10.1371/journal.pone.0009422.g001 translational blocker, it did not elicit the reduction of total Hsp60 level (Fig. S1B). However, the transfection of AS-ODNs indeed selectively reduced cytosolic Hsp60 levels compared to the mock or control S-ODN without affecting the mitochondrial level (Fig. 4A). To understand this phenomenon, we hypothesized that the half-life of Hsp60 protein in two compartments may differ. To prove it, the half-life of cytosol-targeted Hsp60 (Hsp60c) was assessed after inhibition of protein synthesis. Surprisingly, the level of cytosolic Hsp60 protein was rapidly reduced (calculated tK = 3.2 min), while total level of endogenous Hsp60 and IKKa proteins was unchanged (Fig. 4B). Moreover, this reduction was completely blocked by the treatment of a proteasome inhibitor MG132 (Fig. 4C). It is noted that the MG132 treatment also resulted in the remarkable increase of the basal level of the Hsp60c protein. Thus, this result, at least in part, explains why the level of cytosolic Hsp60 was more sensitive to AS-ODN treatment, and it additionally suggests that the level of cytosolic Hsp60 might be controlled by proteasome. The TNF-a-induced IKK/NF-kB activation was then exam- ined in the AS-ODN-transfected cells. An in vitro kinase assay showed that the transfection of AS-ODNs appreciably reduced the IKK activation in response to TNF-a by 60% compared to that of the mock or S-ODN (Fig. 4D). However, the AS-ODNs had no effect on the MAP kinase activation in response to TNF-a (Fig. 4D and Fig. S1C), revealing the specific effect of the Hsp60 AS-ODNs on IKK activation. Furthermore, the AS-ODNs almost completely abolished the NF-kB transcriptional activation in response to TNF-a, whereas S-ODN did not, compared to mock-treated cells (Fig. 4E). Owing to its knockdown efficacy, AS-1 is more potent that AS-2. However, the transfection of ODNs itself did not induce basal NF-kB activation, indicating no off-target effect of ODNs. In addition, the reductive effect of AS-ODNs on NF-kB transcrip- tional activity was also evident in 293T and A549 cells (Fig. S1D). An additional control experiment showed that the AS-ODNs had no affect other transcription factor activation, such as AP-1, NF- AT, and CRE (Fig. S1E). PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9422