Briefly, after washing with cell lysis buffer and kinase buffer, the immunoprecipitates were incubated with a GSK-3 fusion protein substrate and ATP for 30 min at 30°C.
After 5 min at room temperature, the immunoprecipitates were incubated with the reaction buffer (20 mM HEPES (pH 7.5), 20 mM MgCl2, 100 μM ATP containing, 10 μCi/ATP(P)) for an additional 10 min at 37°C.
Thereafter immunoprecipitates were incubated with 30 μl CIP buffer containing 5 units of CIP in the presence or absence of 0.2 mM Na3VO4 phosphatase inhibitor and 7mM EDTA.
Immunoprecipitates were incubated in 30 μl of the mTOR kinase buffer containing 300 ng inactive Akt1 and 500 μM ATP at 37°C.
For kinase reactions, immunoprecipitates were incubated in a final volume of 40 µl for 20 min at 37°C in the mTORC1 kinase buffer containing 500 ng 4EBP1 fusion protein (Santa Cruz Biotechnology) and 500 µM ATP.
Immunoprecipitates were incubated with protein G Sepharose beads (GE Healthcare) for 4 hr at 4°C and then washed 3× with RIPA buffer supplemented with protease inhibitors.
Immunoprecipitates were incubated with a constitutively active Akt recombinant protein in the presence of γP32ATP.
The immunoprecipitates were incubated with the labeled peptide overnight at room temperature.
The immunoprecipitate was incubated at 37 °C for 1 h in kinase reaction buffer containing [γ-P] ATP (80 μCi mL−1; Amersham Bioscience, Little Chalfont, Buckinghamshire, UK) and a recombinant IKK substrate peptide (Upstate Biotechonology, Charlottesville, VA, USA).
The resulting immunoprecipitate was incubated with pure histone-H3 protein in the presence of ATP and kinase buffer.
