Finally, samples were observed with a Tecnai G2 Spirit Twin transmission electron microscope (FEI Co., USA) and a JEM ARM 1300S high-voltage electron microscope (JEOL, Japan). Subcellular fractionation The subcellular fractions for immunoprecipitation were ac- quired by differential centrifugation. Briefly, the HeLa cells (26107 cells) were harvested, rinsed twice with ice-cold PBS, and resuspended in 1 ml of a homogenization buffer (20 mM HEPES (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 2 mM MgCl2, 25 mM KCl, 1 mM AEBSF, 1 mM Na3VO4, 5 mM NaF, 5 mg/ml aprotinin, and 5 mg/ml leupeptin) containing 0.25 M sucrose. After rupturing the cells using a glass Dounce homogenizer, the post-nuclear supernatants were obtained from the homogenate by centrifugation at 750 g for 10 min. The supernatants were separated into pellets (mitochondrial fraction) and supernatants (cytosol fraction) by centrifugation at 15,000 g for 15 min. For ODN transfected cells, the subcellular fractions were obtained using the ProteoExtract subcellular proteome extraction kit (Roche). The purity of each fraction was verified by selective markers: a-tubulin as a cytosolic marker, inner membrane protein cytochrome c oxidase 4 (COX4) and matrix protein peroxiredoxin III (Prx III) [70] as mitochondrial markers. For best comparison, the mitochondrial fraction was loaded at a volume of one-fifth of the corresponding cytosolic fraction. In vitro binding assay with recombinant proteins The Sf9 insect cells were infected with each of recombinant baculovirus stocks harboring IKKa-, IKKb-, and IKKc- encoding bacmids. The insect lysates expressing (His)6-tagged IKKs were incubated with 1.0 mg of GST-Hsp60 proteins pre-bound to glutathione-Spharose beads (Amersham Pharmacia Biotech) at 4uC for 2 hrs. The beads were washed again three times with a cold lysis buffer A. The proteins bound to the beads were eluted by boiling in a SDS sample buffer and then subjected to immunoblot analyses as indicated in Fig. 1c. Transfection The ODNs (200 nM, unless indicated) was transfected for 24 hrs using OligofectamineTM reagent (Invitrogen, USA). The plasmid transfection was achieved using Fugene-6 reagent (Roche, USA). The antibody was tranduced using a ChariotTM protein delivery kit (Active Motif Co., USA), according to the manufac- turer’s instruction. Immunoprecipitation and in vitro kinase assay The HeLa cells were treated with or without TNF-a (10 ng/ml) for the indicated time periods, rinsed once with cold PBS, and lysed in the lysis buffer A. The cell lysates were precleared with 10 ml of protein A/G agarose beads (Amersham Biosciences) for 1 hr. The cleared lysates were incubated with 2 mg of Hsp60, IKKa, IKKb, or IKKc antibodies for 3 hrs and mixed with 20 ml of protein A/G agarose beads. The lysates were further rotated overnight at 4uC. The beads were washed three times with 1 ml of lysis buffer A. The final protein precipitates were subjected to immunoblot analyses. The immune complexes were visualized by using an enhanced chemiluminescence kit (Amersham Biosciences, USA). For the in vitro kinase assay, the IKK, JNK1 or ASK-1 was immunoprecipitated with anti-IKKc (FL-419) or anti-JNK1 (C-17) or anti-ASK-1 (H-300) antibody, respectively. The beads contain- ing the IKK complex or JNK1 were washed twice with lysis buffer and further twice with a kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 10 mM b-glycerolphosphate, 1 mM Na3VO4, Hsp60 for IKK Regulation 2 mM NaF, and 1 mM dithiothreitol), and then incubated in 40 ml of a kinase buffer containing 10 mM ATP, 0.6 mCi [c-32P] ATP, and 2 mg of either GST-IkB (1–54) or GST-c-Jun or GST- SEK1 (K129R) at 30uC for 30 min. The reaction was stopped by adding 20 ml of 36SDS sample buffer. After boiling, the half of reaction mixture was resolved on a 10% denaturing gel and the radioactivity was detected by autoradiography. The other half of reaction mixture was used for immunoblotting of the immuno- precipitated kinase proteins (note: anti-ASK-1 antibody (F-9) for detecting ASK-1). Measurement of Intracellular ROS Intracellular ROS generation was assessed with an oxidation sensitive fluoresce dye, 5,6-chloromethyl-29, 79-dichlorodihydro- fluorescein diacetate (CM-H2DCFDA, Molecular Probes, USA) as described [71]. The HeLa cells (36105) were plated on 35-mm dishes and transfected with ODNs for 24 hrs. The cells were then deprived of serum for 6 hrs and stimulated with TNF-a in phenol red-free media for the indicated periods of time. After stimulation, the cells were quickly rinsed with Krebs-Ringer solution and incubated for 5 min with 5 mM CM-H2DCFDA. The DCF fluorescence was collected for 10 seconds with an inverted Axiovert200 fluorescence microscope (Zeiss). The relative DCF fluorescence was obtained by averaging the fluorescence intensities of the 60–80 cells in each image using ImageQuantTM software (GE Healthcare). Note that the detached round cells were omitted from quantification. RNase protection assay ODN-pretreated HeLa cells were treated with or without TNF- a (10 ng/ml) for the indicated times. The total RNA was extracted with Trizol (Invitrogen). The ribonuclease (RNase) protection assay was performed according to the manufacturer’s protocol (BD PharMingen). Briefly, the human apoptosis template set hAPO-5 was labeled with [a-32P]-uridine triphosphate. The RNA (10 mg) and 66105 cpm of the labeled probes were subjected to hybridization. After the RNase treatments, the protected probes were resolved on 5% urea-polyacrylamide gel and detected by autoradiography. Quantitative PCR (qPCR) Total RNA was extracted using Trizol reagent (Invitrogen) from the HeLa cells stimulated with TNF-a for indicated periods of time. RNA (1.5 mg) was reverse transcribed using ImProm-II RT system (Promega). The real-time PCR was performed using specific primers in the presence of SYBR Green (Applied Biosystems) inside a fluorescent temperature cycler (ABI Prism 7000 sequence detection system, Applied Biosystems). The fluorescence signals were quantified by a comparative cycle threshold method. The actin mRNA was used for an endogenous control. Transgenic mice generation The HA-tagged human Hsp60c lacking mitochondrial signal sequence was PCR-amplified and subcloned into pCAGGS transgenic (Tg) vector, which contains the chicken b-actin promoter, using NheI and EcoRV sites. The HA-Hsp60c Tg construct was linearized by digestion with SalI and PstI and then microinjected into eggs from C57BL/6j females. The transgenic founders were genotyped as described below. Two of the six positive transgenic lines, designated T4 and T11, were chosen for this study. The tail DNA was used for genotyping. In brief, mouse tails were incubated overnight in 100 mM Tris, pH 8.0, 0.5 mM PLoS ONE | www.plosone.org 13 March 2010 | Volume 5 | Issue 3 | e9422