Nbd c6 hpc

Cells were plated and cultured under basal condition with 10% FBS or under starvation with 0.1% FBS overnight, resuspended in lysis buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM CaCl₂, 5 mM MgCl, 5% Glycerol, 0.1% NP-40 and protease inhibitors) and then sonicated. Lysates were clarified using centrifugation and cellular ATG4B activity was assessed by incubation of equivalent lysate amounts with various concentrations of LC3B-PLA2 fusion protein substrate (25–890 nM) in reaction buffer (20 mM Tris-HCl, pH 8.0, 2 mM CaCl and 20 μM NBD-C₆-HPC; Invitrogen). Fluorescence intensity was measured for 2 h at 2-min intervals (60 measurements per data point) using a SpectraMax 5 M plate reader (Molecular Devices) at room temperature with excitation and emission wavelength of 485 nm and 530 nm, respectively. To assess catalytic efficiency, the k_cat/K_m of Atg4B on LC3B-PLA2 was determined using nonlinear regression fitting of a series of progress curves captured at different substrate concentrations using the established sequential activation model34 (link). P values for differences between velocity at each substrate concentration were determined by multiple-measures analysis of variance (ANOVA). Thioredoxin activity was assessed using the insulin disulfide reduction assay36 (link)58 (link).