3. S. J. Baker and E. P. Reddy,Oncogene 12, 1 (1996);

14. HA-p46SAPKg-pCDNA3 is a cytomegalovirus (CMV) promoter–based expression vector encoding a hem-agglutinin (HA)–tagged version of a 46-kD splice vari-ant of SAPKg (11). TRAF expression vectors have been described (5, 9). 293 cells were transfected by calcium phosphate coprecipitation with 8 mg of plas-mid DNA. Forty-eight hours after transfection, cells were lysed in RIPA buffer containing 0.5 mM dithio-threitol (DTT), 20 mM b-glycerophosphate, 1 mM so-dium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, leupeptin (20 mg/ml), and aprotinin (20 mg/ml). Lysates were cleared by centrifugation, and protein concentration was mea-sured by use of a commercial Bradford protein assay (Promega). Equal amounts of each lysate (usually 50