GSK650394

SGK1 S422D (60–431 aa; 0.275 μg/mL final concentration) or SGK2 (0.875 μg/mL final concentration) are activated by PDK1 (1.1 μg/mL final concentration) in a buffer consisting of 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM EDTA, 10 mM MgCl₂, 0.1% β-mercaptoethanol, 1 mg/mL BSA, and ATP (final concentration of 0.15 mM) and incubated for 30 min at 30°C. SGK2 is prepared exactly as described for SGK1, except it corresponded to the full-length protein. A solution containing biotinylated CROSStide peptide at a final concentration of 75 μM and γ³²P-ATP corresponding to 2×10⁶ cpm is prepared in the reaction buffer. In a 96-well plate, 5 μL of GSK650394 is added to 25 μL of the activated enzyme mixture. To this, 20 μL of the CROSStide mixture is added and incubated for 1 h at room temperature. Next, 50 μL of a 25 mg/mL slurry of streptavidin-coated SPA beads in PBS with 0.1 M EDTA, pH 8.0 is added. The plate is then sealed and centrifuged for 8 min at 2000 rpm, and the signal is detected by measuring for 30 sec/well in a Packard TopCount NXT Scintillation Counter. The IC50 values of the inhibition of SGK1 and SGK2 activities by GSK650394 are calculated from these data using GraphPad Prism 3 Software.

LNCaP cells

~10 μM

7 days

LNCaP cells are plated at a density of 5,000 cells per well in 96-well plates in 100 μL PRF-RPMI 1640, supplemented with 8% CS-FBS, 0.1 mM NEAA, and 1 mM NaPyr. At day three, cells are treated with hormone with or without GSK650394 by removing 50 μL of the media and replacing this with 50 μL of PRF-RPMI 1640 with 8% CS-FBS, NEAA, NaPyr containing a 2X concentration of the appropriate hormone/inhibitor treatment. At days 5 and 7, the treatment is repeated. On the tenth day, the media is removed and the relative cell number is measured using the FluoReporter Blue assay according to the manufacturer’s instructions.