Bacterially expressed recombinant GST-NEDD4.2 [DU 8073] was phosphorylated in vitro by PKB, SGK1 and PKA. 50 ng of non-phosphorylated and phosphorylated GST- NEDD4.2 was subjected to SDS-Page analysis, transferred to nitrocellulose membrane and immunoblotted with anti-NEDD4.2 phospho Ser 221 at 1 μg/ml in the presence of 10 μg/ml of the non-phosphorylated peptide. Binding of the primary antibody was detected using donkey anti-sheep IRDye800 (1 in 5000 dilution) followed by visualisation using the LiCor Odyssey system.

In vivo stimulations: HEK293 cells were transfected with an expression vector for GST- NEDD4.2 [DU 3826]. After 36 hours, the cells were deprived of serum for 16 hours and pre-treated with or without 10 μM H89 for 1 hour before being stimulated with or without 10 % FBS for 15 mins, 20 ng/ml IGF-1 for 15 mins and 15 μM forskolin for 30 mins. The cells were lysed and 20 μg of lysate was subjected to SDS/Page followed by transfer to nitrocellulose membrane. In vitro stimulations: Bacterially expressed recombinant GST-NEDD4.2 [DU 8073] was phosphorylated in vitro with SGK1. 50 ng of non-phosphorylated and phosphorylated GST-NEDD4.2 was subjected to SDS-Page analysis with transfer to nitrocellulose membrane. The membranes were immunoblotted with anti-NEDD4.2 phospho Ser 221 at 1 μg/ml in the presence of 10 μg/ml of the non-phosphorylated peptide. Binding of the primary antibody was detected using donkey anti-sheep IRDye800 (1 in 5000 dilution) followed by visualisation using the LiCor Odyssey system.