PrP<sup>Sc</sup> levels in the lysosomal fraction are reduced following autophagy in starved cells.
2015-09-14T02:50:21Z (GMT) by
<p>(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 PrP<sup>Sc</sup> degradation in the isolated lysosomes, PrP<sup>Sc</sup> 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).</p>