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In vitro transcription of capped mRNA with modified nucleotides and Poly(A) tail
TSA (Tyramide Signal Amplification), used for signal amplification of ISH, IHC and IC etc.
Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
A convenient and sensitive way for cell proliferation assay and cytotoxicity assay
Protect the integrity of proteins from multiple proteases and phosphatases for different applications.
HTH-01-015 is a highly specific inhibitor of NUAK1 with IC50 value of 100 nM 1.
HTH-01-015 showed extreme selectivity. It only inhibited the NUAK1 (NUAK family SNF1-like kinase-1) isoform of NUAK kinases and showed no significant inhibition of 139 other tested kinases. HTH-01-015 inhibited the phosphorylation of the NUAK1 substrate, MYPT1. The phosphorylated site was identified as Ser445. In HEK cells overexpressing drug-resistant NUAK1, HTH-01-015 displayed no more inhibition effect on MYPT1. In MEF cells, HTH-01-015 markedly reduced cell migration in the wound-healing assay. Besides that, HTH-01-015 impaired the proliferation at concentration of 10 μM both in U2OS cells and in MEF cells. Moreover, 10 μM HTH-01-015 also significantly inhibited the invasiveness of U2OS cells in a 3D matrigel transwell invasion assay 1.
References:1. Sourav B, Sara J B, Hai-Tsang H, et al. Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. Biochemical Journal, 2014, 457(1): 215-225.
Kinase activity assays
In vitro activities of purified GST–NUAK1 and GST–NUAK1 [A195T] were measured using Cerenkov counting of incorporation of radioactive 32P from [γ -32P] ATP into Sakamototide substrate peptide. Reactions were carried out in a 50 μl reaction volume for 30 min at 30 μC and reactions were terminated by spotting 40 μl of the reaction mix on to P81 paper and immediately immersing in 50 mM orthophosphoric acid. Samples were washed three times in 50 mMorthophosphoric acid followed by a single acetone rinse and air drying. The kinase-mediated incorporation of [γ -32P] ATP into Sakamototide was quantified by Cerenkov counting. One unit of activity was defined as that which catalyzed the incorporation of 1 nmol of [32P]phosphate into the substrate over 1 h.
Cell lines
HEK293, MEFs and U2OS
Preparation method
Limited solubility. General tips for obtaining a higher concentration: Please warm the tube at 37°C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20°C for several months.
Reaction Conditions
37°C
Applications
In HEK-293 cells expressing wild-type NUAK1, 3–10 μM HTH-01-015 significantly inhibits phosphorylation of MYPT1. Treatment of NUAK1+/+ MEFs with 10 μM HTH-01-015 prominently suppresses cell migration in the wound-healing assay. In U2OS cells, HTH-01-015 blocks proliferation and phosphorylation of MYPT1 to the same extent as shRNA-mediated NUAK1 knockdown.
References:
1. Sourav B, Sara J B, Hai-Tsang H, et al. Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. Biochemical Journal, 2014, 457(1): 215-225.
