IRAK: A kinase associated with the interleukin-1 receptor

Science

Volumn 271, Issue 5252, 1996, Pages 1128-1131

  Cao, Zhaodan ^a   Henzel, William J ^b   Gao, Xiong ^a  

^a AMGEN INC   (United States)

^b GENENTECH INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; IMMUNOGLOBULIN ENHANCER BINDING PROTEIN; INTERLEUKIN 1; INTERLEUKIN 1 RECEPTOR; PROTEIN KINASE;

AMINO ACID SEQUENCE; ARTICLE; EMBRYO; HELA CELL; HUMAN; HUMAN CELL; KIDNEY CELL; MOLECULAR CLONING; PRIORITY JOURNAL; SIGNAL TRANSDUCTION; TRANSCRIPTION REGULATION;

EID: 0029865142     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5252.1128     Document Type: Article

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26. 32P]ATP)]. The phosphorylation reaction was incubated at 30°C for 15 min and was then incubated with unlabeled ATP (100 μM) for 15 min. The protein A beads were collected and washed sequentially with 150 ml of wash buffer 1, 150 ml of wash buffer 2 [50 mM Hepes (pH 7.9), 1 M NaCl, 5 mM DTT, 1 mM EDTA, and 0.1% NP-40] and 150 ml of wash buffer 3 [50 mM Hepes (pH 7.9), 100 mM NaCl, 2 M urea, 5 mM DTT, 1 mM EDTA, and 0.1% NP-40)]. Proteins that remained bound were eluted with 50 ml of the elution buffer (buffer 3 with 7 M urea) overnight at 4°C with rocking. The eluted material was loaded onto a 0 5-ml Q Sepharose column. After they were washed extensively with the elution buffer, proteins bound (including pp100) were eluted with 1.5 ml of the elution buffer with 0.5 M NaCl. The eluate was concentrated in a Microcon 50 (Amicon) to 50 μl, diluted with 1 ml of isoelectrofocusing sample buffer (15), concentrated again to 50 μl, and then subjected to 2D gel electrophoresis.
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39. Cells were collected by centrifugation in 5 ml of phosphate-buffered saline with 1 mM EDTA, washed once with medium (10 ml), sedimented, resuspended in 1 ml of medium, and transferred to 1.5-ml microtubes. IL-1β (200 ng/ml) was added to the tubes, followed by incubation at 37°C for the indicated time. The cells were collected by centrifugation and then lysed with 1 ml of lysis buffer (14). After incubation on ice for 30 min, the cell debris was sedimented in a microcentrifuge and discarded. The IL-1RI complexes were immunoprecipitated (12), resolved by SDS-PAGE, and transferred to nitrocellulose membrane, which were blotted with antiserum to IRAK.
40. 2-terminal regions of IRAK and Pelle, A. Ashkenazi for providing IL-1 RI expression plasmids; A. Bothwell for pNeoSRαII, K. Williamson for nucleotide sequencing; L Xu and S. Wong for technical support; and V. Balchwal, M. Rothe, and U. Schindler for critical review of the manuscript.