Figure 4

Manipulation of ARTD10 levels reflects on GSK3β activity in cells. A. GFP- ARTD10 or GFP-ARTD10-G888W was co-expressed with HA-GSK3β in U2OS cells. Immunoprecipitated HA-GSK3β was subsequently subjected to a kinase assay with a primed substrate peptide and [³²P]-γ-ATP. Incorporated radioactivity was measured by scintillation counting. B. Western blot analysis of the ARTD10 and GSK3β expression and immunoprecipitation levels of the kinase tested in (A) C. U2OS cells were transfected with siARTD10 or siControl SMARTpools, GSK3β was immunoprecipitated and subjected to kinase assays with a primed substrate peptide and [³²P]-γ-ATP. Incorporated radioactivity was measured by scintillation counting. D. Western blot analysis of the ARTD10 and GSK3β expression levels of the kinase tested in (C) E. U2OS cells were transfected with HA-ARTD10, HA-ARTD10-G888W, HA-GSK3β or ARTD10 shRNA. 48 hours after transfection cells were lysed and analyzed.