Fig. 5

Phosphorylated NSF exhibits enhanced ATPase activity. a NSF ATPase activity was assessed with a Malachite green assays at 36nM NSF and increasing concentrations of ATP substrate (up to 1.4 mM) in the presence of NSF alone, NSF pre-phosphorylated by LRRK2-G2019S^970–2527 (ΔG2019S) or LRRK2-G2019S^970–2527 (ΔG2019S) alone (NSF:LRRK2 20:1). Data were fitted with the Michaelis-Menten kinetic model to determine kinetic constants. b Phosphate generated by ATP hydrolysis in the presence of NSF wild-type, NSF^T645A NSF^T646A NSF^S647A pre-phosphorylated or not by ΔG2019S was measured with the Malachite Green Assay at 120 min with an initial concentration of ATP of 1.4 mM (NSF^WT vs NSF^T645A **p < 0.01; NSF^WT vs P-NSF^WT ***p < 0.001; NSF^T645A vs P-NSF^T645A p > 0.05, non significant, n.s.; NSF^T646A vs P-NSF^T646A p > 0.05, n.s.; NSF^S647A vs P-NSF^S647A **p < 0.01; one-way ANOVA, Bonferroni’s post-test, n ≥ 3). UT = untrasfected cells subjected to Flag-affinity purification to monitor background activity (n = 1; excluded from the statistical analysis)