Methods

Cells and Reagents
Jurkat E6.1, HEK293T and HeLa cells were cultured in RPMI 1640 and DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin, respectively. IκBα (C-21, sc-371), p65 (C-20, sc-372), and phospho-threonine (H-2, sc-5267) antibodies were from Santa Cruz Biotechnology; β-actin (AC-15, A5441), Flag (M2, F3165), HA (HA-7, H3663), importin-α (IM-75, I1784), and importin-β (31H4, I2534) antibodies were from Sigma; PARP (C2-10, 556362), IKKα (B78-1, 556532), and IKKβ (24, 611254) antibodies were from BD Pharmingen; CK2α (31, 611610) and Hsp90 (68, 610418) antibodies were from BD Transduction Laboratories; phospho-IκBα (5A5, 9246S) and phospho-IKKα/β (16A6, 2697S) antibodies were from Cell Signaling Technology; phospho-serine (AB1603) and phospho-tyrosine (4G10, 05-777) antibodies were from Millipore. The rabbit polyclonal RPS3 antiserum was as described previously6. The rabbit polyclonal antibody specific for S209 phosphorylated RPS3 was generated and affinity purified by Primm Biotech using the peptide NH2-CKPLPDHV(Sp)IVE-COOH.

Plasmid Constructs
The Flag-IKKβ (SSEE), Flag-IKKβ (SSAA), and HA-IκBα (SSAA) constructs were provided by C. Wu (NCI, Bethesda) and U. Siebenlist (NIAID, Bethesda), respectively. The HA-IκBα and IKKβ (K44A)-Flag plasmids were purchased from Addgene44, 45. The Flag-RPS3, GST-RPS3, HA-RPS3, VN-HA, NleH1-HA plasmids were described previously6, 9. The point mutants of RPS3 were generated by site-directed mutagenesis using the Quick Change Kit (Stratagene) with primers forward 5′-CTGCCTGACCACGTGGCCATTGTGGAACCCAAA-3′ and reverse 5′-TTTGGGTTCCACAATGGCCACGTGGTCAGGCAG-3′ for S209A. All mutants were verified by DNA sequencing.

32P in vivo Labeling
HEK 293T cells were labeled with 2 mCi/ml 32P-orthophosphate (Perkin Elmer) in phosphate-free medium (Invitrogen) for 2 h. Cells were then left untreated or treated with TNF (50 ng/ml, R&D Systems) for indicated periods. Cell lysates were prepared and used for immunoprecipitations with RPS3 antibody.

In Vitro Kinase Assay
Kinase-active recombinant IKKβ and IKKα proteins were purchased from Active Motif and Millipore, respectively. Bacterially purified glutathione S-transferase (GST), GST-IκBα (1-54), wild type, mutant GST-RPS3, or RPS3 proteins were used as substrates. The in vitro kinase assay was performed as previously described29. Briefly, enzyme (100 ng) and substrate (2 μg) were co-incubated in IKK reaction buffer (25 mM Tris–HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, 1 mM ATP) or NleH1 reaction buffer (50 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 1 mM DTT, 1 mM ATP) with 0.5 μCi 32P-γ-ATP (GE Healthcare) added at 37 °C for 30 min. The reactions were resolved by SDS–PAGE and visualized by autoradiography.

LC-MS/MS Analysis
GST or GST-RPS3 was incubated with recombinant IKKβ protein as described above in an in vitro kinase assay reaction conducted without 32P-γ-ATP labeling. The reaction was separated by SDS-PAGE, and the protein gel was stained with Colloidal Blue (Invitrogen). The corresponding protein fragments were excised and subjected to trypsin digestion and LC-MS/MS at the Yale Cancer Center Mass Spectrometry Resource (New Haven, CT).

RNAi and Transfection
The siRNA (sense-strand sequence) IKKα, 5′-AUGACAGAGAAUGAUCAUGUUCUGC -3′; IKKβ, 5′-GCAGCAAGGAGAACAGAGGUUAAUA -3′; IκBα, 5′-GAGCUCCGAGACUUUCGAGGAAAUA -3′; RPS3-3′ UTR, 5′-GGAUGUUGCUCUCUAAAGACC -3′ (Invitrogen). Transient transfection of siRNA and DNA constructs into Jurkat cells and 293T cells was described previously6.

Subcellular Fractionation
Subcellular fractionation was performed by differential centrifugation as previously described6. Briefly, cells were resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.4 % NP-40, 0.5 mM PMSF, complete protease inhibitor cocktail) at 4 °C for 5 min. Lysates were centrifuged at 4 °C, 500 × g for 3 min, and supernatants were collected as cytosolic fractions. Pellets were incubated in Buffer C (20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, complete protease inhibitor cocktail) at 4 °C for 10 min. Supernatants were collected as nuclear fractions following a centrifuge at 4 °C, 13,800 × g for 10 min.

Luciferase Reporter Gene Assays
Luciferase reporter gene assays were performed as previously described6. Briefly, cells were cotransfected at a ratio of 10:1 with various promoter-driven firefly luciferase constructs to the Renilla luciferase pTKRL plasmid, together with indicated plasmids. Cells were cultured for 1–2 days and then stimulated in triplicate before harvest. Lysates were analyzed using the Dual-Luciferase Kit (Promega).

Chromatin Immunoprecipitation (ChIP)
ChIP assays was performed as previously described6. The primers used to amplify the promoter region adjacent to the κB sites of IL8 and NFKBIA, as well as ACTB have been described6.

Immunofluorescence Microscopy
Confocal microscopy was performed as previously described6. Briefly, cells were fixed with 4 % paraformaldehyde in PBS and then Cellspin mounted onto slides. The fixed cells were then permeabilized with 0.05 % Triton X-100 in PBS and stained with FITC-conjugated rabbit anti-RPS3 antibodies (Primm Biotech), or AlexaFluor 594-conjugated rat anti-Flag antibodies (BD) for 40 min together with 1 μg/ml of Hoechst 33342 (Sigma) for 5 min at 25 °C. The slides were then rinsed with PBS three times and cover mounted for fluorescence microscopy.

Immunoprecipitation and immunoblot
The cells were harvested and lysed on ice by 0.4 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) supplemented with 1 × protease inhibitor cocktail (Roche) and 1 × phosphatase inhibitor cocktail set I (EMD Biosciences) for 30 min. The lysates were centrifuged at 10,000 × g at 4 °C for 10 min to remove insoluble material. After normalizing protein concentrations, lysates were subjected to immunoprecipitation by adding 10 mg/ml appropriate antibody plus 30 ml of protein G-agarose (Roche), and rotated for at least 2 h at 4°C. The precipitates were washed at least five times with cold lysis buffer followed by separation by SDS-PAGE under reduced and denaturing conditions. Nitrocellulose membranes were blocked in 5 % nonfat milk in 0.1 % PBS-Tween 20 (PBS-T), probed with specific antibodies as described previously6. For immunoblotting of phosphorylated proteins, gels were transferred to methanol-treated polyvinylidene chloride membranes, retreated with methanol, and dried for 30 min. Blots were blocked in 5 % bovine serum albumin in 0.1 % Tris buffered saline-Tween 20 (TBS-T), and probed with specific antibodies as described previously46. Bands were imaged by the Super Signaling system (Pierce) according to the manufacturer's instructions.

ELISA
The amount of IL-8 present in supernatants collected from Jurkat cell culture was measured using a Human Interleukin-8 ELISA Ready-SET-Go kit (eBioscience) according to the manufacturer's instructions.

Cell Infections
HeLa cells were infected with E. coli O157:H7 or C. rodentium strains as described previously9.

Immunohistochemistry
Gnotobiotic piglets were infected with E. coli O157:H7 strains as described previously9. Spiral colon specimens were collected at necropsy and embedded in paraffin. Paraffin sectioning and immunohistochemical staining using phospho-RPS3 antibody were performed by Histoserv Inc.