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. 2015 Aug 4;11(8):e1005073. doi: 10.1371/journal.ppat.1005073

Fig 4. ROCK inhibition lowers PrPSc level by rescuing TACE α-secretase neuroprotective activity towards PrPC in a PDK1-dependent manner.

Fig 4

(A, B) Amount of PrPSc in Rocki-Fk-1C115-HT 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. (C) Immunofluorescent labeling of TACE at the surface of Fk-infected 1C115-HT cells treated or not with the ROCK inhibitor (Y-27632, 100 μM) for 1 h. Scale bar, 50 μm. (D) Immunoblot analysis of sucrose gradient fractions of membranes of Fk-infected 1C115-HT cells treated or not with Y-27632 (100 μM, 1h) versus uninfected 1C115-HT to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles. FAK = Focal Adhesion Kinase. Cav-1 = caveolin-1. (E) Immunofluorescent labeling of TACE at the surface of Rocki-Fk-1C115-HT differentiated for 4 days in the presence of Y-27632 (100 μM) versus uninfected 1C115-HT cells. Scale bar, 50 μm. (F) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in Fk-infected 1C115-HT 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. (G) PDK1 activity in Fk-infected 1C115-HT cells treated or not with Y-27632 (100 μM, 1 h), in Fk-infected 1C115-HT bombarded with tungsten microprojectiles coated with ROCK-I or ROCK-II antibodies or both, or in Rocki-Fk-1C115-HT cells differentiated for 4 days in the presence of Y-27632 (100 μM) versus uninfected 1C115-HT cells. n = 5 for each condition. Values are the mean ± s.e.m. for all experiments. * P <0.01 versus uninfected cells. ** P <0.01 versus non-treated infected cells. # P <0.05 versus uninfected cells. ## P <0.05 versus non treated infected cells. ### P <0.05 versus infected cells treated with Y-27632.