NAIA

Application of NAIA. HepG2 cells were incubated with NAIA (10 µM) in complete medium for 60 min. The cells were then washed with PBS, fixed by 4% paraformaldehyde, permeabilized, and reacted with azide-fluor 545 (20 µM) by CuAAC reaction at room temperature in the dark for 1 h. The stained cells were washed with PBS and further incubated with Hoechst (8.2 µM) for 15 min. The cells were then washed with PBS and imaged by confocal fluorescence microscopy.

Application for NAIA. HepG2 cells were treated with solvent vehicle, H₂O₂ (1 mM) or H₂O₂ (1 mM) + NAC (5 mM) in a serum-free medium for 15 min at 37 °C. The cells were then incubated with NAI (50 µM; 10 min) to label-free thiols in live cells, followed by cell lysis and TCEP (1 mM) reduction for 1 h. After acetone precipitation at -20 °C overnight, the proteins were labeled by NAIA-5 (100 µM) in PBS. The samples were then prepared for MS and analyzed by LC-MS/MS.

Application for NAIA in Gel-based ABPP experiment. HepG2 cell lysates (100 µg) in PBS were incubated with NAIA for 60 min, followed by copper-catalyzed azide-alkyne cycloaddition reaction with azide-fluor 545 (25 µM). The protein mixtures were then boiled with sampling buffer, resolved by SDS-PAGE and imaged.