Enzyme Kinetic Assay: PTP activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate in 3,3-dimethylglutarate buffer (50 mM 3,3-dimethylglutarate, pH 7.0, 1 mM EDTA, 150 mM NaCl, 2 mM DTT, 0.1 mg/mL BSA) at 25° C. The assays were performed in 96-well plates. Normally, to determine the IC50 values, the reaction was initiated by the addition of enzyme (final concentration at 10 nM) to a reaction mixture (0.2 mL) containing 2 mM (Km for the substrate) pNPP with various concentrations of inhibitors. The reaction rate was measured using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices). To determine the mode of inhibition, the reactions were initiated by the addition of PTP-MEG2 to the reaction mixtures (0.2 mL) containing various concentrations of pNPP with different concentrations of the inhibitor 7.
Inhibition of recombinant human cytosolic PTPepsilon expressed in Escherichia coli BL21 (DE3) using E527-P-Q-pY530-Q-P-G-E-N-L536 as substrate after 60 mins by malachite green assay
Inhibition of cytosolic PTPepsilon in human monocyte-derived osteoclasts assessed as relative level of Tyr530 phosphorylated c-Src/beta actin at 17 uM after 15 mins in presence of M-CSF/RANKL by Western blot method relative to control
Inhibition of cytosolic PTPepsilon in human monocyte-derived osteoclasts assessed as disruption of podosome organization at 17 uM after 4 days in presence of M-CSF/RANKL by DAPI staining-based immunofluorescence assay
Inhibition of human PTPRE (107 to 707 residues) expressed in Escherichia coli strain BL21(DE3) using DiFMUP as substrate preincubated for 10 mins followed by substrate addition and measured after 10 mins by fluorescence assay
Inhibition of PTPep (unknown origin) using DiFMUP as substrate incubated for 30 mins followed by substrate addition at pH 6.5 by standard phosphatase assay based fluorescence analysis
Inhibition of PTPep (unknown origin) using DiFMUP as substrate at 100 uM incubated for 30 mins followed by substrate addition at pH 6.5 by standard phosphatase assay based fluorescence analysis
Inhibition of PTPE (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
Inhibition of His-tagged PTPepsilon (unknown origin) expressed in Escherichia coli BL21 cells using para-nitrophenyl phosphate as substrate incubated for 5 to 10 mins by microplate reader based spectrophotometric analysis