Phosphor storage plate

Wild-type and T493M RSK2 kinase activity were assayed as reported previously ^{26 (link)}. Briefly, ERK2-activated RSK2 CTD (5 nM) was incubated with varying concentrations of each inhibitor for 30 minutes in the presence of 100 μM ATP and 10 mM GSH. Each reaction was initiated by the addition of 167 μM substrate peptide (RRQLFRGFSFVAK) and 0.3μCi/μL γ-³²P-ATP in a final volume of 25 μL for an additional 30 minutes. Reactions were spotted on phosphocellulose membranes, washed once with 10% AcOH, twice with 0.1% H₃PO₄, and once with MeOH prior to drying. Blots were exposed to a phosphor storage plate (Amersham Biosciences), imaged with a Typhoon scanner (GE Healthcare), and quantitated using the SPOT program ^{60 (link)}. IC₅₀ values were calculated using a sigmoidal dose response fitting in the Prism program (Graphpad) and are reported as the average of two biological experiments. Full IC₅₀ curves are presented in Supplementary Fig. 17.

JAK3 kinase activity was assayed using recombinant JAK3 (Invitrogen, catalog # PV5774). JAK3 (3.1 nM) was incubated with varying concentrations of each inhibitor for 30 minutes in the presence of 11.5 μM ATP. Each reaction was initiated by the addition of 17.9 μM substrate peptide and 0.3 μCi/μL γ-³²P-ATP in a final volume of 25 μL for an additional 60 minutes. Reactions were spotted on phosphocellulose membranes, washed once with 10% AcOH, twice with 0.1% H₃PO₄, and once with MeOH prior to drying. Blots were exposed to a phosphor storage plate (Amersham Biosciences), imaged with a Typhoon scanner (GE Healthcare), and quantitated using the SPOT program^{60 (link)}.