Fig. 2. Trypsin proteolysis of neurofilaments isolated from control (WT) and NFHLacZ transgenic (TG) mice. (A) Neurofilaments (25 µg) were incubated during 20 minutes with 20 ng (for NFM) or 4ng (for NFH and NFL) of trypsin at 30°C. The reaction was stopped by the addition of Laemmli buffer, boiled during 5 minutes and analyzed by western blot. (B) 50 µg of neurofilaments were digested for 20 minutes at 37°C with trypsin (2 ng for NFH and NFL, and 4 ng for NFM), then sedimented for 30 minutes at 100,000 g and 4°C. Supernatant (S) and pellet (P) fractions were analysed by western blot. Similar proteolytic patterns and solubility of fragments were observed between control and transgenic mice.