RSK2 in vitro phosphorylation, immunoblot analysis and activity assay

For activation of RSK2, 4.8 μM samples of RSK2 were incubated in 0.05 μM of PDK1 and 0.047 μM of ERK (final concentration) in 20 mM Tris buffer containing 500 mM NaCl, 10 mM of MgCl2, and 200 μM of ATP at room temperature for 2 hrs. Aliquots of activation reaction were taken at different time points and analyzed for site-specific phosphorylation by immunoblot analysis. (Phosphorylation by either PDK1 or ERK alone, reported in Supplementary data was carried out in the same fashion).

Samples (~50 ng) of RSK2 were resolved on polyacrylamide gels, transferred to Immobilion R-FL polyvinylidine difluoride membrane (Millipore), which were then blocked with the Odyssey Blocking Buffer (TBS) LI-COR. The membranes were subjected to phospho-RSK2 (Ser 227, Ser 386, Thr 365/Ser 369, Thr 577) and total RSK-2 antibodies diluted in blocking odyssey buffer (1:8000). Next the membranes were washed 3 times in TBS with 0.05% Tween 20. Primary antibodies were visualized using fluorophore-labeled secondary antibodies conjugated to either Alexa Fluor® 680 or IRDye®800CW, and scanned with an Odyssey infrared imaging system (LI-COR Biosciences).

Activated RSK2 was assessed for enzymatic activity at room temperature using 0.2 mg/ml (160 μM) of S6 peptide and either 10 or 100 μM of ATP in a kinase buffer (40 mM Tris, 20 mM MgCl₂). The enzymatic activity of RSK2 was determined by measuring the amount of ATP consumed in the reaction using ADP-Glo system (Life Technologies) and a luminescence plate reader (PHERAstar FS, BMG Labtech). In some experiments SL0101 at concentrations ranging between 0.1 and 100 μM was added to the reaction.