His6 senp1 catalytic domain

Measurements were performed using six different inhibitor concentrations starting at 200 μM of 11 with three-fold dilution. Human recombinant, His6-SENP1 Catalytic Domain (Boston Biochem Cat# E-700, Bio-Techne AG, Zug, Switzerland) was prepared in reaction buffer (50 mM HEPES, 150 mM NaCl 0.5 mM EDTA, 1 mM DTT, pH 7.5, 0.01% CHAPS) as 555 nM solution. A 20 mM compound DMSO stock-solution (1.1 µL) was added to the freshly prepared 555 nM enzyme solution (98.9 µL). Five sequential three-fold dilutions resulted in a final inhibitor concentration of 0.8 µM and the enzyme inhibitor complex was incubated for 15 min at 0 °C. Then, 50 µM proSUMO3 (5 µL) was added to the freshly prepared enzyme inhibitor solution (45 µL) and incubated for 60 min at 37 °C and subsequently the reaction was stopped by adding 4 × Laemmli-Buffer (16.7 µL) and denaturing the protein at 100 °C for 30 min. Those solutions (10 µL) were loaded on a casted 15% sodium dodecyl sulfate polyacrylamide gel and separated on a Mini-PROTEAN Tetra Cell (Bio-Rad, Hercules, CA, USA). After SDS-PAGE gel electrophoresis separation, the gels were stained with staining solution Bio-Safe Coomassie Brilliant Blue G-250 (BioRad) for 60 min. De-staining was performed with 40% (v/v) methanol, 10% (v/v) glacial acetic acid, 50% (v/v) Milli-Q water until protein bands became visible.

IC₅₀ measurements were performed with at least two replications using eight concentrations starting at 200 μM and for the fragments at 500 μM with three-fold dilution. Human recombinant, His6-SENP1 Catalytic Domain (Boston Biochem Cat# E-700, Bio-Techne AG, Zug, Switzerland) was prepared in reaction buffer (50 mM HEPES, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, pH 7.5, 0.01% CHAPS). To this solution, a 20 mM or 50 mM inhibitor stock solution in DMSO was added and the mixture was incubated for 15 min at ambient temperature. The substrate SUMO1/2 or 3-AMC (SUMO1-AMC; Boston Biochem Cat# UL-551; SUMO2-AMC; Boston Biochem Cat# UL-758; SUMO3-AMC; Boston Biochem Cat# UL-768; Bio-Techne AG, Zug, Switzerland) was delivered to a 96 well-plate to initiate the reaction. The enzyme activities were monitored (Ex/Em = 355/460 nm) as a time-course measurement of the increase in fluorescence signal from SUMO1/2 or 3-AMC for 60 min at 25 °C. The data were analyzed by taking the slope (signal/time) of the linear portion of measurement. The slope was calculated using Excel and the curve fits were performed using GraphPad Prism7 with a four-parameter least-squares fit. The final reaction conditions contained 0.02 nM SENP1 and 300 nM SUMO1/2 or 3-AMC).