antibody was obtained from Qiagen; Antibodies to phosphor- IKK, IKKa, and IKKb were from Cell Signaling Technology; Normal mouse and rabbit IgG were from Amersharm Bioscience; Anti-cytochrome c antibody were from BD Pharmingen; Anti- bodies to Peroxiredoxin III (Prx III), MnSOD (2AI), Hemagglu- tinin epitope (HA), and GAPDH were provided by AbFrontier (Seoul, Korea); Recombinant human TNF-a was purchased from Invitrogen (Grand Island, USA). The phosphorothioate oligodeox- ynucleotides (ODNs), including the antisense and sense sequences, were synthesized by Hokkaido System Sciences Co. (Hokkaido, Japan). The full-length human IkB protein was a kind gift of W. Jeong (Ewha Womans University, Korea) [64]. Plasmids The full-length cDNA of human Hsp60 was obtained from the National Genome Information Center (Daejon, Korea). A truncated form of Hsp60, designated as Hsp60c, which lacks a mitochondrial targeting sequence (MTS; amino acids 1–26 based on human sequence), was amplified by PCR and subcloned into the pCGN-HA (a kind gift of Dr. W. Herr, Cold Spring Harbor Laboratory) and pGEX-4T1 (Amersham) vectors, by which the HA-tagged and GST-fused Hsp60c expression plasmids, respec- tively, were constructed. CRE-, NF-AT-, and AP1-dependent (pAP17x-Luc) firefly luciferase reporters were obtained from Stratagene. Luciferase reporter plasmids harboring the IFNb-derived NF-kB enhancer sequences [65] was a kind gift of S. Y. Lee (Ewha Womans University, Korea). Human IKK a, b, and c cDNAs [66,67] were subcloned into the pCMV2-FLAG or baculovirus expression vector pFastBac-HTa (Invitrogen). The pFastBac contructs, encoding each of IKKa, b, and c, were used for production of high-titer recombinant baculovirus stocks (,16107 pfu/ml), according to the manufacturer’s protocol. The pPuro plasmids encoding human Bcl-2 or Bcl-XL were kindly provided by D.Y. Shin (Dankook University, Korea) [68]. The plasmid pGEX-4T1- SEK1 (K129R) [69] was used for production of GST- SEK1(K129R) recombinant protein. Site-directed mutagenesis was performed using a QuikChange mutagenesis kit (Stratagene). Immuno-affinity purification of IKK complex and ESI-q- TOF tandem mass spectrometry HeLa S3 cells (20 ml packing volume from 20-l suspension culture) were gently lysed in 200 ml of lysis buffer A (20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM AEBSF, 1 mM Na3VO4, 5 mM NaF, 10 ug/ml aprotinin and leupeptin). The lysate (2 grams of total protein) was precleared with agarose beads alone for 1 hr, and then incubated overnight with anti-IKKa-conjugated agarose beads (2 mg/ml IgG, Santa Cruz Biotechnology). After extensively washing four times with the lysis buffer, the beads were loaded onto a column and rinsed twice with phosphate-buffered saline. The precipitated proteins were eluted twice with 1 ml of 0.1 M glycine buffer (pH 2.5). The protein eluates were immediately neutralized by adding 1 M Tris HCl buffer (pH 8.0) and separated on a 10% denaturing gel. The gel was subsequently stained with silver nitrate, and the silver-stained spots were subjected to in-gel trypsin digestion with minor modifications as follows. Briefly, the gel spots were excised with a scalpel and destained by washing with 15 mM K4Fe(CN)6, 50 mM sodium thiosulfate. The gel pieces were crushed, dehydrated by adding acetonitrile, rehydrated by adding 10–20 ml of 25 mM ammonium bicarbonate with 10 ng/ml of sequencing grade trypsin (Promega), and incubated at 37uC for 15–17 h. The peptides in the supernatant were transferred to a new tube and extracted twice Hsp60 for IKK Regulation by adding 50 ml of a solution containing 60% acetonitrile and 0.1% trifluoroacetic acid. The extracted solutions were pooled and evaporated to dryness in a SpeedVac vacuum centrifuge. The tandem mass spectral (MS/MS) analysis for peptide sequencing was done with nano flow reversed-phased HPLC/ESI/MS with a mass spectrometer (Q-TOF UltimaTM global, Waters Co. UK). Peptides were separated by using a C18 reversed-phase 75mm i. d.6150 mm analytical column (3 mm particle size, AtlantisTM dC18, Waters) with an integrated electrospray ionization Silica- TipTM (610 mm, New Objective, USA). In detail, 5ml of peptide mixtures were dissolved in buffer A (water/ACN/formic acid; 95:5:0.2, v/v), injected onto a column and eluted by a linear gradient of 5–80% buffer B (water/ACN/formic acid; 5:95:0.2, v/ v) over 120 min. Samples were desalted on a line prior to separation using a trap column (i.d. 0.35650 mm, OPTI-PAKTM C18, Waters) cartridge. Initially, the flow rate was set to 200 nL/ min by a split/splitless inlet and the capillary voltage (3.0 keV) was applied to the HPLC mobile phase before spraying. Chromatog- raphy was performed online using the instrument’s control software MassLinx of Q-TOF UltimaTM global. The mass spectrometer was programmed to record scan cycles composed of one MS scan followed by MS/MS scans of the eight most abundant ions in each MS scan. MS parameters for efficient data- dependent acquisition were intensity (.10) and number of components (3,4) to be switched from an MS to MS/MS analysis. Following positive identification, all identified peptides from database search (Mascot) were excluded in the next run analysis until full sequence coverage was obtained. Database analyses using the database search programs including Mascot (global search engine), Proteinlynx 2.1 (Waters Co., UK) and MODi (Korea, http://modi.uos.ac.kr/modi/), provided almost full sequence coverage on selective exclusion monitoring. MS/MS spectra were matched against amino acid sequences in SwissProt. Precusor ion mass corrections and a fragment ion mass tolerance of 0.2 Da were used to consider as 2 missed cleavages. Immunoelectron microscopy The HeLa cells (16107 cells) were harvested and fixed for 1 hr, at room temperature in 0.1 M cacodylate buffer (pH 7.2) that contained 0.5% glutaraldehyde. After rinsing with cold distilled water, they were dehydrated through an ethanol treatment series at 4uC. They were infiltrated with LR White resin (London Resin, Berkshire, England) at 4uC and embedded in LR White resin in a gelatin capsules (Nisshin EM, Tokyo, Japan). Polymerization of the resin was carried out at 50uC for 24 hrs. Serial sections (120–200 sections per one sample), 70 nm in thickness, were attached to formvar-coated nickel grids. Sections were incubated in 50mM glycine for 5 min at room temperature. After rinsing with PBS, sections were incubated in 3% BSA for 30 mins at room temperature. Then, they were incubated with primary antibodies (goat anti-human Hsp60 (SC-1722), mouse anti-human IKKa (SC-7606), mouse anti-IKKb (SC-8014), diluted 1:100 in PBS) for 2 hrs, at room temperature. After washing five times with Tween- PBS (PBS plus 0.5% Tween-20), sections were treated with 20 nm- and 40 nm-diameter colloidal gold conjugated to anti-goat and anti-mouse IgG + IgM antibodies, respectively (BB International, UK, diluted 1:20 in PBS) for 2 hrs, at room temperature. The sections were washed three times with Tween-PBS and then washed three times with distilled water. Sections were stained with 4% uranyl acetate for 5 mins and with lead citrate for 5 min. To examine the specificity of the primary antibody, a treatment of sections was performed with the same procedure without of the primary antibody. For double staining, antibody reactions were repeated with the second set of primary and secondary antibodies. PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9422