10.1371/journal.ppat.1005073.g004

ROCK inhibition lowers PrP<sup>Sc</sup> level by rescuing TACE α-secretase neuroprotective activity towards PrP<sup>C</sup> in a PDK1-dependent manner.

Abstract

<p>(<b>A, B</b>) Amount of PrP<sup>Sc</sup> in Rocki-Fk-1C11<sup>5-HT</sup> cells differentiated into serotonergic neuronal cells for 4 days (4d) in the presence of Y-27632 (100 μM) or 22L-infected CGNs exposed for 6 h to Y-27632 (100 μM), as shown by western blotting (left) and quantified by ELISA (right). n = 10 for each condition. (<b>C</b>) Immunofluorescent labeling of TACE at the surface of Fk-infected 1C11<sup>5-HT</sup> cells treated or not with the ROCK inhibitor (Y-27632, 100 μM) for 1 h. Scale bar, 50 μm. (<b>D</b>) Immunoblot analysis of sucrose gradient fractions of membranes of Fk-infected 1C11<sup>5-HT</sup> cells treated or not with Y-27632 (100 μM, 1h) versus uninfected 1C11<sup>5-HT</sup> to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles. FAK = Focal Adhesion Kinase. Cav-1 = caveolin-1. (<b>E</b>) Immunofluorescent labeling of TACE at the surface of Rocki-Fk-1C11<sup>5-HT</sup> differentiated for 4 days in the presence of Y-27632 (100 μM) versus uninfected 1C11<sup>5-HT</sup> cells. Scale bar, 50 μm. (<b>F</b>) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in Fk-infected 1C11<sup>5-HT</sup> cells treated or not with Y-27632 (100 μM) or a combination of Y-27632 (100 μM) and TAPI-2 (100 μM) for 6 h versus uninfected cells and the ratio (bottom) of C1/native full-length PrP. n = 5 for each condition. (<b>G</b>) PDK1 activity in Fk-infected 1C11<sup>5-HT</sup> cells treated or not with Y-27632 (100 μM, 1 h), in Fk-infected 1C11<sup>5-HT</sup> bombarded with tungsten microprojectiles coated with ROCK-I or ROCK-II antibodies or both, or in Rocki-Fk-1C11<sup>5-HT</sup> cells differentiated for 4 days in the presence of Y-27632 (100 μM) versus uninfected 1C11<sup>5-HT</sup> cells. n = 5 for each condition. Values are the mean ± s.e.m. for all experiments. * <i>P</i> <0.01 versus uninfected cells. ** <i>P</i> <0.01 versus non-treated infected cells. # <i>P</i> <0.05 versus uninfected cells. ## <i>P</i> <0.05 versus non treated infected cells. ### <i>P</i> <0.05 versus infected cells treated with Y-27632.</p

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oai:figshare.com:article/1502384Last time updated on 2/12/2018

This paper was published in FigShare.

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