Takinib

Kinase experiment:

Activity of purified TAK1-TAB1 protein is measured. In brief, TAK1-TAB1 (50 ng/well) is incubated with 5 μM ATP containing radiolabeled [32P]-ATP in the presence of 300 mM substrate peptide (RLGRDKYKTLRQIRQ) in a final volume of 40 μL in the presence of buffer (containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-Mercaptoethanol, 10 mM magnesium acetate, 0.5 mM MnCl) and indicated compounds. The reaction is let go for 10 min and stopped with 10 μL concentrated H3PO4. The remaining activity is measured using a scintillation counter. Dose-response curves are repeated 3 times. For kinetic mechanistic studies, experiments are repeated two times and averaged[1].

Cell experiment:

MDA-MB-231 cells (1,000 cells/well) are seeded in a 96-well plate with 10% FBS, 5% Pen/Strep, 4 g/L glucose DMEM medium. After 24h, cells are serum starved with 1% FBS, 5% Pen/Strep, 4 g/L glucose DMEM medium for 4h. Cells are treated with titrations of Takinib in the presence or absence of 30 ng/mL TNFα. Plates at 0 h and 24 h following treatment are frozen at -80°C after removal of media. After 24 h, 100 μL ddH2O is added to each well and plates are refrozen. 1 μL from Hoechst stock [1 mg/mL in 1:4 DMSO/H2O] is dissolved in 1 mL of TNE buffer (10 mM Tris, 2 M NaCl, 1 mM Na2EDTA) and 100 μL of this solution is added to each well. The fluorescence is determined at 355/460 nm[1].

References:

[1]. Totzke J, et al. Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease. Cell Chem Biol. 2017 Aug 17;24(8):1029-1039.
[2]. Totzke J, et al. Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease. Cell Chem Biol. 2017 Aug 17;24(8):1029-1039.e7.