Protein tyrosine kinase substrate LAT and its use in the indentification of (ant)agonists of the kinase

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art.
Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.
g.
, electroporation, lipofection).
Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications.
The techniques and procedures are generally performed according to conventional methods in the art and various general references (see, generally, Sambrook et al.
Molecular Cloning: A Laboratory Manual, 2d ed.
(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.
Y.
, and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc.
, N.
Y.
) which is incorporated herein by reference) which are provided throughout this document.
All the information contained therein is incorporated herein by reference.
Oligonucleotides can be synthesized on an Applied BioSystems oligonucleotide synthesizer [for details see Sinha et al.
, Nucleic Acids Res.
12:4539 (1984)], according to specifications provided by the manufacturer.
Complementary oligonucleotides are annealed by heating them to 90.
degree.
C.
in a solution of 10 mM Tris-HCl buffer (pH 8.
0) containing NaCl (200 mM) and then allowing them to cool slowly to room temperature.
For binding and turnover assays, duplex DNA is purified from native polyacrylamide (15% w/v) gels.
The band corresponding to double-stranded DNA is excised and soaked overnight in 0.
30 M sodium acetate buffer (pH 5.
0) containing EDTA (1 mM).
After soaking, the supernatant is extracted with phenol/chloroform (1/1 v/v) and precipitated with ethanol.
DNA substrates are radiolabeled on their 5'-OH group by treatment with [g-.
sup.
32P]ATP and T4 polynucleotide kinase.
Salts and unincorporated nucleotides are removed by chromatography on Sephadex G columns