Antibody used for immunoprecipitation | Treatment | Histone phosphorylation | Kinase renaturation |
---|---|---|---|
% of respective control | |||
Anti-pp60s r c | Control | 100 ± 18 | 100 ± 21 |
10 nm TCDD | 220 ± 382-a | 238 ± 592-b | |
Nonspecific IgG | Control | 100 ± 16 | No detectable activity |
10 nm TCDD | 98 ± 16 | No detectable activity |
Identical aliquots of C57 liver cytosol were incubated with 10 nm TCDD or p-dioxane only (control) for 10 min at 25° as described in Materials and Methods. After treatment, pp60s r c was immunoprecipitated as described in Materials and Methods. The immune complex was washed extensively and then resuspended in 50 ml of kinase buffer containing 10 mmTris, pH 7.5, 5 mm MnCl2, 3 mmunlabeled ATP, 2 mCi [γ-32P]ATP, and 8 mg of histone/reaction. After a 15 min reaction at 25°, two 20-ml aliquots of the reaction mix was spotted onto two phosphocellulose discs. After extensive washing of the phosphocellulose discs, the remaining radioactivity was counted in a liquid scintillation counter. The control values for the pp60s r c immune complexes were ∼3-fold greater than the control values for the nonspecific immune complexes (nonspecific IgG); values are mean ± standard deviation from three experiments. Relative quantitative scanning using AMBIS (refer to Fig. 6 for a representative gel); values are mean ± standard deviation of four experiments.