Mpo fluorometric activity assay kit

ACS indicator probe APF was purchased from ThermoFisher Scientific (Carlsbad, CA). Sodium hypochlorite, sodium orthovanadate (SOV, PTP inhibitor), hMPO from human leukocytes, and MPO fluorometric activity assay kit were purchased from Sigma-Aldrich (St. Louis, MO). H₂O₂ was purchased from Fisher Scientific (Hampton, NH). HOCl concentration was documented spectrophotometrically in 10 mM NaOH at pH 12 using the molar extinction coefficient at 292 nm (ε_{292 =} 350 M⁻¹ cm⁻¹). Dulbecco’s phosphate buffered saline (DPBS 1X, 21–031-CV) without calcium and magnesium was purchased from Mediatech Inc. (Manassas, VA). Halt phosphatase inhibitor cocktail (PTP + PSTP inhibitors) was purchased from ThermoFisher Scientific (Waltham, MA). Okadaic acid (PSTP inhibitor) was purchased from MilliporeSigma (Burlington, MA). Amplex Red hydrogen peroxide/peroxidase assay kit was purchased from ThermoFisher Scientific (Carlsbad, CA). Synthetic SDG (LGM2605) was purchased from Chemveda Life Sciences Pvt. Ltd. (Hyderabad, India) based on procedure developed by our group. Structures of SDG and protein phosphatase inhibitors are shown in Supplemental Figure 1.

MPO activity was determined using MPO fluorometric activity assay kit purchased from Sigma-Aldrich (St. Louis, MO). In this assay, MPO catalyzes the formation of hypochlorous acid which reacts with the substrate, aminophenyl fluorescein, to generate fluorescein (λ_ex= 485/λ_em=525nm). One unit of MPO is the amount of enzyme that will oxidize the MPO substrate to yield 1.0 umole of fluorescein per min at room temperature. Assay was performed according to the instruction provided in the manufacture’s manual.