Antibodies and Reagents. Monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich (St. Louis, MO). Linear polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA, USA). PTP inhibitor V was purchased from Merck KGaA (Darmstadt, Germany).
Cell Culture and Transfection. HEK 293 cells were cultured in DMEM (Thermo Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific) and 1% penicillin/streptomycin in a 5% CO2 incubator. The day before transfection, 1 × 106 cells were plated in 60 mm cell culture dish and were allowed to adhere overnight. The cells were transfected with FLAG-DUSP13B using PEI.
Purification of Recombinant Protein. His-tagged DUSP13B was constructed in pET28a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into BL21 (DE3)-RIL Escherichia coli. Recombinant protein was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 3 h. Cells were harvested and then lysed by sonication in 50 mM Tris-HCl (pH 6.8), 300 mM NaCl, 1% IGEPAL CA- 630 (NP-40), 1 mM phenylmethanesulfonyl fluoride (PMSF). The lysates were clarified by centrifugation at 10,000 rpm for 20 min at 4 °C. The supernatant was applied to a column filled with Ni-NTA resin (PEPTRON, Daejeon, Korea). The resin was washed with the buffer containing 20 mM Tris- HCl (pH 8.0), 500 mM NaCl, 50 mM imidazole and eluted with the buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200 mM imidazole.
In vitro Phosphatase Assays and Kinetic Analysis. Phosphatase activity was measured by using OMFP (Sigma- Aldrich) as a substrate in a 96-well microtiter plate. The assay was based on methods described previously.12 PTP inhibitor V and OMFP were solubilized in dimethyl sulfoxide (DMSO). All reactions were performed at the final concentration of 1% DMSO. The final reaction mixture (100 μL) was optimized for enzyme activity and composed of 30 mM Tris-HCl (pH 7.0), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), 0.33% bovine serum albumin (BSA) and 100 nM of PTPs. Reactions were initiated by addition of OMFP and incubated for 30 min at 37 °C. Fluorescence emission from the product was measured by a multi-well plate reader (Biotek, excitation filter, 485 nm; emission filter, 535 nm). The reaction was linear over the time period of the experiment and was directly proportional to both enzyme and substrate concentrations. IC50 value was defined as the concentration of an inhibitor that caused a 50% decrease in the activity of PTP. IC50 values and the best curve fit for Lineweaver-Burk plots were determined by using the curve fitting program Prism 3.0 (GraphPad Software, San Diego, CA). All experiments were performed in triplicate and repeated at least three times.
Immunoblotting Analysis. Cell lysates were run in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose membrane (Whatman, Springfield Mill, UK). The membrane was blocked with 5% skim milk for 1 h and incubated with the appropriate primary antibodies, followed by incubation with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP). The protein bands were visualized by the ECL detection system (Pierce, Rockford, IL).
Immunoprecipitation and in vitro Phosphatase Activity Assay. HEK 293 cells were transiently transfected with FLAGDUSP13B expression plasmid. After 48 h of incubation, cells were washed with phosphate buffered saline (pH 7.4) to remove the remaining inhibitor on the cells. Then the cells were lysed with PTP lysis buffer containing 0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM PMSF and 3 μM DTT for 15 min at 4 °C. Cell lysates were clarified by centrifugation at 13,000 rpm for 30 min at 4 °C and the supernatant were incubated with anti-FLAG M2 affinity gel on a rotator for 3 h at 4 °C. After binding, the beads were washed with lysis buffer and the bound proteins were incubated with various concentrations of PTP inhibitor V and OMFP. The reaction buffer was comprised of 30 mM Tris-HCl (pH 7.0), 75 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 0.33% BSA. The reaction mixtures were incubated at 37 °C for 30 min and the fluorescence were measured by multi-well plate reader (excitation filter, 485 nm; emission filter, 535 nm).
Inhibition Study. The inhibition constant (Ki) to DUSP13B for the inhibitor was determined by measuring the initial rates at several OMFP concentrations for each fixed concentration of the inhibitor. The data were fitted to the following equation to obtain the Ki value of reversible competitive inhibitors. The obtained slopes were replotted against the inhibitor concentrations. The Ki value was obtained from the slopes of these replots.13
1/V = Km (1 + [I]/Ki) Vmax [S] + 1/Vmax