PrPSc levels in the lysosomal fraction are reduced following autophagy in starved cells.
(A) Lysosomes were isolated by the fractionation of N2a-FK cells incubated or not in HBSS for 8 h, as an inducer of autophagy. PK-treated or-untreated gradient fractions were applied at one-tenth of their original volume per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP, anti-Lamp1 (as a lysosomal marker) and anti-β-actin (as the internal standard) antibodies. Fractions 1 to 3 contained high-quality, purified native lysosomes. Positive controls consisted of cell lysates containing 15 μg of protein before purification (P). (B) To quantify ratio of the PrPSc degradation in the isolated lysosomes, PrPSc band intensities were measured as a percentage of those of the controls. Empty columns indicate the control, non-treated fractions and shaded columns the HBSS-treated fractions. The results in the graph are the mean ± SD of at least three independent experiments. *p < 0.05 (Student's t-test).