4.2. PTP1B Enzymatic Assay

In vitro enzymatic activity of PTP1B was measured by using 4-nitrophenyl phosphate disodium salt (pNPP) as substrate. CYC31 and PTP1B_1-321 protein was pre-incubated at room temperature for 5 min. Assay was conducted in a 96-well plate with reaction buffer containing Tris-Hcl (10 mM, pH 7.5), NaCl (25 mM), EDTA (1 mM), and pNPP (5 mM). The reaction mixture was then incubated at 37 °C for 30 min and stopped by adding 50 µL of 3 M NaOH. The absorbance density of p-nitrophenol was measured by a multi-well microplate reader at the wavelength of 405 nm.