Immunoprecipitated GFP-Foxc2 or GFP-Foxc2-S124L constructs were incubated for 60
min at 30°C with and without 500U of purified protein kinase CK2 (New England
Biolabs, Ipswich, MA, USA) in the presence of kinase buffer (New England Biolabs, Ipswich,
MA, USA), 20 μM cold ATP (Sigma, St. Louis, MO, USA) and 2
μCi of [γ-32P]ATP (Perkin
Elmer, Waltham, MA, USA). In vitro kinase reactions were terminated by
addition of SDS-PAGE sample buffer and boiling for 5 min. Samples were subjected to
SDS-PAGE, and the amount of 32P incorporated into GFP-Foxc2 or GFP-Foxc2-S124L
was analyzed by autoradiography, followed by immunoblot analysis.
min at 30°C with and without 500U of purified protein kinase CK2 (New England
Biolabs, Ipswich, MA, USA) in the presence of kinase buffer (New England Biolabs, Ipswich,
MA, USA), 20 μM cold ATP (Sigma, St. Louis, MO, USA) and 2
μCi of [γ-32P]ATP (Perkin
Elmer, Waltham, MA, USA). In vitro kinase reactions were terminated by
addition of SDS-PAGE sample buffer and boiling for 5 min. Samples were subjected to
SDS-PAGE, and the amount of 32P incorporated into GFP-Foxc2 or GFP-Foxc2-S124L
was analyzed by autoradiography, followed by immunoblot analysis.