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1842295012
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note
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4, 1 mM benzamidine, 2 μM PMSF, aprotinin at 10 μg/ml, leupeptin at 1 μg/ml, pepstatin at 1 μg/ml, 1 mM DTT], and the kinase was eluted in a gradient to PS buffer with no ammonium sulfate. Fractions containing IκB kinase activity were subjected to immunoblot analysis with anti-RelA, anti-IκB-β, and anti-MKP-1 (Santa Cruz Biotechnology, Inc.)or anti-MEKK-1 or anti-P-Tyr (Upstate Biotechnology).
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1842416966
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note
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4, 1 mM benzamidine, 2 μM PMSF, aprotinin at 10 μg/ml, leupeptin at 1 μg/ml, pepstatin at 1 μg/ml, 1 mM DTT] at 30°C for 30 to 60 min in the presence of the indicated substrate. The kinase reaction was stopped by addition of 6× SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, subjected to SDS-PAGE analysis, and visualized by autoradiography. GST-IκB substrates were expressed and purified from Escherichia coli with glutathione Sepharose 4B beads. His-tagged RelA-streptavidin-tagged IκB-α WT and His-tagged Rel A-streptavidin-tagged IκB-α (S32A, S36A) mutant complexes were purified from Sf9 cells coinfected with the indicated baculoviral expression constructs.
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1842414671
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unpublished data
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F. Mercurio, B. Bennett, B. Murray, L. Ransone, H. Zhu, A. Manning, unpublished data.
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Mercurio, F.1
Bennett, B.2
Murray, B.3
Ransone, L.4
Zhu, H.5
Manning, A.6
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0031285250
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F. S. Lee, J. Hagler, Z. J. Chen, T. Maniatis, ibid. 88, 213 (1997); M. Hirano et al., J. Biol. Chem. 271, 13234 (1996); N. L. Malinin, M. P. Bolin, A. V. Kovalenko, D. Wallach, Nature 385, 540 (1997).
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F. S. Lee, J. Hagler, Z. J. Chen, T. Maniatis, ibid. 88, 213 (1997); M. Hirano et al., J. Biol. Chem. 271, 13234 (1996); N. L. Malinin, M. P. Bolin, A. V. Kovalenko, D. Wallach, Nature 385, 540 (1997).
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1842299149
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note
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4, 1 mM benzamidine, 2 μM PMSF, aprotinin at 10 μg/ml, leupeptin at 1 μg/ml, pepstatin at 1 μg/ml, 1 mM DTT] or up to 3.5 M urea.
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34
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1842412921
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note
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32P was detected by scintillation counting. Less than 10% of the ATP was consumed in the reaction.
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35
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1842334327
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note
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For large-scale IKK signalsome purification, HeLa S3 cells were stimulated for 7 min with TNF-α at 20 ng/ml (R&D Systems) and harvested; whole-cell lysate was prepared (1.2 g of total protein) and about 5 mg of anti-MKP-1 (Santa Cruz Biotechnology, Inc.) was added and incubated at 4°C for 2 hours with gentle rotation. Then 50 ml of protein A-agarose (Calbiochem) was added and incubated for 2 hours. The immunoprecipitate was then sequentially washed four times with PD buffer, two times with RIPA buffer, two times with PD buffer, once with 3.5 M urea-PD buffer, and three times with PD buffer. The immunoprecipitate was then made into a thick slurry by adding 10 ml of PD buffer and 25 mg of the MKP-1 peptide to which the antibody was generated (Santa Cruz Biotechnology, Inc.); then it was incubated overnight at 4°C with gentle rotation. Salt was removed from the eluted protein on PD10 columns (Pharmacia) equilibrated with 50 mM Q buffer, and eluate was chromatographed on a Mono Q column (Pharmacia). Fractions containing IκB kinase activity were pooled, concentrated, and subjected to preparative SDS-PAGE; protein bands were visualized with colloidal blue stain (Novex) and the bands were excised and sequenced.
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36
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1842295546
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note
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Coomassie blue-stained bands were excised and digested with trypsin as described. A small portion of the supernatant was removed for analysis by MALDI peptide mapping as described (16). The program PeptideSearch (EMBL, Heidelberg) was used to compare the peptide mass map from the IKK-1 band with a protein sequence database. Eight measured peptide masses matched those calculated for peptides from CHUK within 30 ppm (18). The peptide mass map of the IKK-2 band did not result in a clear identification and therefore the sample was subject-ed to nanoelectrospray mass spectrometry (25). The peptide mixture was micropurified on a capillary containing 50 nl of Poros R2 resin (Perseptive Biosystems, Framingham, MA). The peptides were washed and then step-eluted with 0.5 μl of 50% MeOH in 5% formic acid into a nanoelectrospray needle. This needle was transferred to an APIII mass spectrometer (Perkin-Elmer, Sciex, Toronto, Canada) and the sample was sprayed for about 20 min. During this time, peptide ions apparent from the mass spectrum were selected and isolated and then fragmented in the collision chamber of the mass spectrometer. From the tandem mass spectra, short stretches of sequence were assembled into peptide sequence tags (18) and compared with a protein sequence database or an EST database by using PeptideSearch. Three peptides matched the IKK-1 sequence. A1, IIDLGYAK; A2, VEVALSNIK; A3, SIQLDLER. Three other peptides matched human EST sequences in the EST database: B1, ALELLPK; B2, VIYTQLSK; B6, LLLQAIQSFEK. These three sequences all match EST clone AA326115. The peptide B4 with the sequence LGTGGFGNVIR was found in clone R06591. After the full-length IKK-2 sequence was obtained (19), two more peptides (B3, ALDDILNLK; B5, DLK-PENIVLQQGEQR) were found in the sequence. Peptide A1 is present in both IKK-1 and IKK-2 sequences.
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0028575316
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1842330157
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note
-
Several peptides were identical matches to human EST clones. All the EST clones were similar to human and mouse CHUK-1 (IKK-1). These clones were obtained (Genome Systems, Inc.) and the precise nucleotide sequence was determined and used to design primers to clone human IKK-2 by polymerase chain reaction (PCR) from a human HeLa cell cDNA library (Clontech, Inc.). Several IKK-2 cDNA clones were isolated and sequenced. Full-length mouse IKK-1 and a partial human IKK-1 nucleotide sequence were available in the comprehensive database. Primers were designed to clone by PCR the human and mouse IKK-1 cDNAs. The partial human IKK-1 coding region was used to probe a HeLa cDNA phage library (Stratagene) to obtain the full-length human IKK-1 cDNA clone by standard procedures.
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43
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1842404602
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note
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For in vitro translation studies, HA-tagged IKK-1 and Flag-tagged IKK-2 were in vitro translated in RRLs, either separately or alone, exactly as described in the manufacturer's protocol (Promega).
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45
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1842374898
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note
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For immunocytochemistry, HeLa cells were transiently transfected with either HA-tagged IKK-1 or Flagtagged IKK-2 as indicated. Cells were washed with PBS, fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 30 min, and permeabilized with wash buffer (0.5% Triton X-100, 0.01 % azide in PBS). Cells were blocked with wash buffer containing donkey serum (5%) (Jackson lmmunoresearch Laboratories) and probed with primary antibody-anti-RelA (polyclonal antibody), 1:2000 (Santa Cruz); anti-HA (monoclonal antibody), 1:1000 (Eastman Kodak); or anti-Flag (monoclonal antibody), 1:1000 (Babco)-followed by secondary antibody-donkey antibody to rabbit (fluorescein isothiocyanate) conjugate, 1:100; donkey antibody to mouse (Texas Red conjugate), 1:100 (Jackson Immunoresearch Laboratories). Cover slips were mounted with polyvinyl alcohol and 1,4-diazabicyclo[2.2.2]octane (Sigma) mounting medium and the slides were viewed under fluorescence with a Nikon Microphot-FXA microscope; the images were then scored and photographed.
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note
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We thank I. Verma and R. Mueller for the gift of the IκB-α COOH-terminus construct and helpful discussions; J. DiDonato for the IκB-α vectors; H. Raymon and N. Richard for assistance with immunocytochemistry; S. Kim, A. LaPointe, and B. Lee for technical assistance; K. Davis and E. Carlson for help with compiling the manuscript and figures; and D. Anderson and A. Lewis for helpful comments and support. A.R. contributed to this work during a sabbatical at Signal Pharmaceuticals.
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