Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells

Science

Volumn 271, Issue 5253, 1996, Pages 1272-1276

  Li, Wei ^a   Whaley, Carmella D ^a   Mondino, Anna ^a   Mueller, Daniel L ^a  

^a UNIVERSITY OF MINNESOTA MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CALCINEURIN; CD28 ANTIGEN; CD3 ANTIGEN; INTERLEUKIN 2; LYMPHOCYTE ANTIGEN RECEPTOR; NUCLEAR FACTOR; PHORBOL 13 ACETATE 12 MYRISTATE; PROTEIN KINASE;

ANIMAL CELL; ARTICLE; CLONAL ANERGY; DEPHOSPHORYLATION; MOUSE; NONHUMAN; PRIORITY JOURNAL; SIGNAL TRANSDUCTION; T LYMPHOCYTE; T LYMPHOCYTE ACTIVATION;

ANIMALIA; MURINAE;

EID: 0029975023     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5253.1272     Document Type: Article

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57. A protein immunoblot analysis of cellular proteins was also used to examine ERK and JNK protein abundance. Approximately 20 μg of whole-cell extract (33) was mixed 1:1 with 2× SDS sample buffer, and then separated by 10% SDS-PAGE. Proteins were transferred to nitrocellulose, and blotted as described (34) with either polyclonal antibody 691 to ERK-1 and ERK-2 at a 1:1000 dilution or with polyclonal antibody to JNK-1 (1 μg/ml) (Santa Cruz Biotechnology).
58. 32P into the fusion protein during the first 15 min was linear with respect to both time and kinase concentration The immobilized proteins were washed three times with kinase wash buffer, eluted in 1× SDS sample buffer, and then analyzed on a 10% SDS-polyacrylamide gel, followed by autoradiography to detect the presence of a phosphorylated GST-c-Jun substrate. The phosphorylated fusion protein electrophoresed through an SDS gel as several bands because of protein degradation
59. We thank G. R Crabtree (plasmids pNFATZH and pSXNeo/AP-1), T. Geppert (rabbit antibodies A249 to ERK-2 and 691 to ERK-1 and ERK-2), and A Rao (rabbit antiserum R59 to recombinant NFATp) for generously providing reagents and expertise critical to the work; T. Behrens and M. Mescher for critically reviewing the manuscript; L. Chiodetti, S. Garcia, and S. Seiffert for expert technical assistance; and M. K. Jenkins for many valuable discussions. Supported by a grant from NIH (R29 AI31669), as well as funding from the Minnesota Arthritis Foundation. A.M. is a recipient of a fellowship from the American-Italian Foundation for Cancer Research. D L.M. is the recipient of an Investigator Award from the National Arthritis Foundation.