Characterization of an immune complex kinase in immunoprecipitates of avian sarcoma virus-transformed fibroblasts
N D Richert, P J Davies, G Jay, I H Pastan
1979
Journal of Virology
Kinase activity detected in immune complexes containing the src gene product of the avian sarcoma virus has been reported. To further characterize this immune complex kinase, we developed a routine quantitative assay involving trichloroace-MATERIALS AND METHODS Celis and viruses. Cultures of chicken embryo fibroblasts (CEF) were prepared from 10-day-old embryonated eggs (Truslow Farms, Chestertown, Md.) by the method of Vogt (34). Primary cultures were infected with cloned viruses from the
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... wing sources: SR-ASV (subgroup D) and RAV-50 were from American Type Culture Collection, Rockville, Md.; the SR-ASV mutants, tsNY68 and tdNY107A, were from H. Hanafusa; BH-ASV(RAV-1) and the Bryan temperature-sen. itive mutant (Ta) were from John Bader. Clones of normal rat kidney cells, SR-ASV (subgroup D)-trforrmed normal rat kidney cells (SR-NRK), and Kirstcn sarcoma virus-transformed normal rat kidney cells were obtained from E. Scolnick. Preparation of antiserum. Antiserum was prepared by injecting newborn rabbits (<1 day old) with approximately 108 focus-forming units of SR-ASV 695 on May 9, 2020 by guest http://jvi.asm.org/ Downloaded from 696 RICHERT ET AL. (subgroup D). The animals were bled at weekly intervals starting at approximately 4 weeks, and all sera were heat inactivated at 56°C for 30 min and clarified by centrifugation at 8,000 x g before use. The antisera were screened for activity in two ways: first, ability to immunoprecipitate kinase activity from SR-CEF (described below) and, second, ability to precipitate p609 from ['5S]methionine-labeled SR-CEF. Preparation of boiled S. aureus. Batch cultures of Formalin-fixed Staphylococcus aureus (strain Cowan I) were prepared by the method of Kessler (13) and stored in 1-ml volumes at 4°C. Within 1 to 2 weeks before use, each volume was centrifuged at 3,000 x g for 5 min. The bacterial pellet was suspended in 1 ml of TBS (0.05 M Tris [pH 7.2]-0.15 M NaCI) containing 10%o (vol/vol) mercaptoethanol and 3% (wt/vol) sodium dodecyl sulfate (SDS) and heated at 95°C for 30 min. After centrifugation, the bacteria were centrifuged and suspended in fresh buffer, and the heating was repeated. The final bacterial pellet was washed twice in RIPA (6) buffer (1% Triton-1% deoxycholate-0.1% SDS-0.5% Trasylol [Sigma] in TBS [pH 7.2]) and suspended in 1 ml of RIPA for the immunoprecipitation procedure. The boiled S. aureus preparation reduces the nonspecific binding of [;5S]methionine-labeled proteins and also reduces nonspecific trichloroacetic acid-precipitable counts in the kinase assay. Preparation of cell extracts. Secondor thirdpassage CEF cultures were used for all experiments. The cells were plated in 100-mm petri dishes, 24 to 48 h before use, at densities of 8 x 106 cells per plate or 5 x 10' cells per plate, respectively. Extracts were prepared by the method of Collett and Erikson (6). Briefly, the monolayers were washed
doi:10.1128/jvi.31.3.696-706.1979
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