Protein kinases assayed at . 1 mM ATP had been RIP2, GAK, c Raf and B Raf. The assays had been initiated with MgATP, stopped by the addition of 5 ul of . 5 M orthophosphoric acid and spotted on to P81 filter plates making use of a unifilter harvester. The ICvalues of inhibitors had been determined following carrying out assays at 10 various concentrations of every compound.

PKA was assayed against the substrate peptide LRRASLG, PKC and GAK from the protein histone H1, PHK towards the substrate peptide KRKQISVRGL, NEK2a from the peptide RFRRSRRMI, NEK6 and NEK7 against the peptide FLAKSFGSPNRAYKK, ROCK and PRK2 towards a peptide corresponding to the Cterminal location of ribosomal protein S6. Aurora B and Aurora C had been equally assayed kinase inhibitor library for screening in opposition to the substrate peptide HIPK3, MST 2, IKK and IKK towards MBP, RIP2 towards MBP, IKKB against the peptide LDDRHDSGLDSMKDEEY, and JNK2 and JNK3 in opposition to ATF2. MARK3 was assayed towards the peptide KKKVSRSGLYRSPSMPENLNRPR, RSK1, RSK2, MAPKAP K3 and PKD1 from KKLNRTLSVA, MNK1 and MNK2 towards the eIF4E protein, EF2K assayed from the peptide RKKFGESKTKTKEFL and PIM1, PIM2 and PIM3 from RSRHSSYPAGT.

PKBB was assayed from the peptide calculator peptide GRPRTSSFAEGKK, PLK1 against ISDELMDATFADQEAKKK, Src towards KVEKIGEGTYGVVYK, CaMK 1 from YLRRRLSDSNF, smMLCK from KKRPQRATSNVFA and SRPK1 against RSRSRSRSRSRSRSR. DYRK1A, DYRK2 and DYRK3 were both assayed in opposition to Woodtide, while PAK4, 5 and 6 had been assayed in opposition to RRRLSFAEPG. CaMKK, CaMKKB and TBK1 were assayed from BRSK2 from KKLNRTLSFAEPG and PKC? towards ERMRPRKRQGSVRRV. The protein tyrosine kinases Indeed, FGF R1 and Ephrin A2 have been assayed with poly. The substrates employed for other protein kinases have been described previously. Unless of course mentioned otherwise, enzymes were diluted in a buffer consisting of 50 mM Tris/HCl, pH 7. 5, . 1 mM EGTA, 1 mg/ml BSA and . 1% 2 mercaptoethanol and assayed in a buffer comprising fifty mM Tris/HCl, pH 7. 5, .

1 mM EGTA and . 1% 2 mercaptoethanol. For CaMK1 and CaMKK isoforms, the assay mixtures also contained . 5 mM CaCland . 3 uM calmodulin. PKC was diluted into twenty mM Hepes /. 03 Triton X one hundred and assayed in the very same buffer containing . 1 mg/ ml phosphatidylserine, 10 ug/ml diacylglycerol and . 1 mM CaCl. PHK was diluted in fifty mM sodium B glycerophosphate /. 1%2 mercaptoethanol VEGF and assayed in a buffer comprising 50 mM Tris/HCl, fifty mM sodium B glycerophosphate, pH 8. 2, and . 04 mM CaCl. EF2K was diluted into 50 mM Hepes /. 1% 2 mercaptoethanol/1. mg/mlBSAand assayed in the identical buffer that contains . 2 mM CaCland . 3uM calmodulin. smMLCK was diluted in fifty mM Hepes /. 1 mM EGTA/1. mg/ml BSA/. 1% 2 mercaptoethanol and assayed in the exact same buffer that contains 5 mM CaCland 10 uM calmodulin.

PKA was Natural items diluted in twenty mM Mops /1 mM EGTA/. 01% Brij 35/1. mg/ml BSA/. 1% 2 mercaptoethanol and assayed in 8 mM Mops /. The compound SU 6656 is noted to be a effective inhibitor of Src household members.

In the present review we identified that it inhibited AMPK, kinase inhibitor library for screening BRSK2 and MST2 with equivalent potency to its inhibition of Src and Lck, and it inhibited Aurora B and C, even more potently than Src and Lck in vitro.