Enzcheck phosphatase assay kit

The phosphatase assay was conducted with the EnzCheck Phosphatase Assay Kit (Molecular Probes, Eugene, OR, USA). The 100 μL reaction mixture in the 96-well black plate for the measurement of phosphatase activity consisted of a 100 μM 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate, 1 mM metal such as Mg²⁺, Zn²⁺, Mn²⁺, or Ca²⁺, 100 mM NaCl, 25 mM Tris-HCl pH 7.0, and a 10 μM protein sample. The reaction was started by adding the protein, and the results were measured with the Filter Max F3 (Molecular Devices, San Jose, CA, USA) as soon as possible after adding the protein to the reaction mixture. The interval of measurement was 3 min. The amount of the product, 6,8-difluoro-4-methylumbelliferyl (DiFMU), in the solution was determined photometrically at 460 nm. The graph for the phosphatase assay was modified using Origin 2022 (OriginLab, Northampton, MA, USA). The data were fitted to the quadratic equation to calculate a Kd value between Mg²⁺ and Sav2152, as follows: $$\mathrm{V}={\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\times \frac{\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\mathrm{K}\mathrm{d}-\sqrt{{\left(\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\mathrm{K}\mathrm{d}\right)}^2-4\times \left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]\times \left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]\times \mathrm{K}\mathrm{d}}}{2\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]}$$
where V represents the initial reaction velocity, V_max represents the maximum initial reaction velocity, P_total represents the total amount of protein, A_total represents the total amount of Mg²⁺ ion, and Kd represents a dissociation constant.

Phosphatase activity was measured using the EnzCheck Phosphatase assay kit (Molecular Probes) in samples without transfection, and those transfected with control DNA, WT PTPN23 and Mutant PTPN23. Lysates were made in Reporter lysis buffer (Promega) and incubated with substrate DiFMUP and measured using an excitation of ~360 nm and an emission detection of 460 nm. Potato Acid Phosphatase, provided by the manufacturer, was used as a control. Each bar is an n = 3 on independent transfections, and each control was an independent dilution of the Potato Acid Phosphatase, n = 3. Results are expressed as a fold increase over untransfected, where values are set to 1.