Buffer components:
25 mM Hepes
10 mM MgCl2
1 mM PEP
280 uM NADH
30 ug/mL Pyruvate kinase (PK)
10 ug/mL Lactate Dehydrogenase (LDH)
2 mM DTT
ATP (see below)
Peptide substrate VSP-1 (see below)
1.5% DMSO
JNK3 (see below)

Assay run as follows:
• 1.5 uL compound/DMSO dispensed to empty assay plate from master titration plates containing 10
• point titrations of 8 compounds, (10 uM-2.6 nM, 2.5 fold dilutions) using Beckman FX
• Use Multidrop to add 80 uL assay buffer to assay plate (Buffer, ATP, and peptide substrate)
• Preincubate plates on warming block 5 minutes to bring up to 30oC
• Use Multidrop to add 20 uL enzyme buffer to assay plate (Buffer and JNK3 at 5x)
• Shake in reader 7 sec, read 6 min at 30oC, cut off first 20 seconds

Kinase: activated JNK3 (bisphosphorylated on the Tyr and Thr in the flexible loop)
[Kinase], nM: 25
[ATP] in assay, uM: 20
Km ATP, uM: 20
Peptide substrate: VSP-1 (sequence: 'KRELVEPLTPSGEAPNQALLR')
[peptide], uM: 200

The enzyme Ki's are derived from 10 point curves.
The majority of the compounds had their Ki values determined only once with a few
performed 2 or 3 times.

Please note, that while activated JNK3 was used in this assay, the crystal structures are of UNACTIVATED JNK3, so the flexible loop is almost certainly in a different conformation.