Fig. 2. (A) Ub-tau species (−254, −311, −353) (5 μM) (B) were incubated with 10 nM h20S in 25 mM Tris–HCl, pH 7.5, 37 °C. The digestion of tau species was monitored over 3 h, until linearity was observed. Proteins harvested at each time-point were heat-denatured, separated by SDS-PAGE and stained by CBB. (C) Band intensity was then calculated by ImageJ software and the rate of substrate cleavage, that is expressed as relative ratio of nmol of substrate left undigested, calculated by subtracting the intensity of the band at the specific time-point in the absence of h20S to that in the presence of h20S. A nominal value 1 was attributed to band intensity at time 0. A representative SDS-PAGE is shown. Data at each time point show the mean ± SD (n = 3 values) computed across independent experiments. ***p ≤ 0.001 significantly different slopes, linear model computed by lmstats package in R.