Fig. 4

VCP inhibition does not affect seed uptake or αS protein levels. A Quantification of percentage of Alexa-647 positive cells (seed uptake) from αS biosensors co-treated with Alexa-647 tagged αS PFF together with vehicle or NMS-873 (1μM) for 4 hours. n=3 biological repeat. Data is mean ±S.E.M. n.s. no significant from student’s t test. B Quantification of seed uptake from αS biosensors co-treated with Alexa-647 tagged αS PFF and LLoMe or vehicle for 4 hours. n=3 biological repeat. Data is mean ±S.E.M. n.s. no significant from student’s t test. C Quantification of seed uptake from VCP or scramble siRNA treated cells with 4-hour Alexa-647 tagged αS PFF application. n=4 biological repeat. Data is mean ±S.E.M. n.s. no significant from student’s t test. D Quantification of seed uptake from αS biosensors treated with Dynogo-4a or DMSO for one hour and then Alexa-647 tagged αS PFF for four hours. n=4 biological repeat. Data is mean ±S.E.M. ****p < 0.0001 from student’s t test. E Immunoblot of anti-Ubiquitin (FK-2), and total α-synuclein (syn-1) from αS biosensors treated for four-hours with NMS-873 (5μM) or DMSO. α-tubulin and HSP90 are loading controls. F Quantitation of band intensities of VCP and α-synuclein in E. *** p < 0.001 and n.s.= no significant by two-way ANOVA. G Immunoblot of anti-VCP, and α-synuclein (syn-1) from αS biosensors treated with scrambled or VCP siRNA for 48 and 72 hours. GAPDH and HSP90 are loading controls. H Quantitation of band intensities of VCP and α-synuclein in G. ** p < 0.01 *** p < 0.001 and n.s.=no significant by two-way ANOVA. I Quantification of CFP fluorescent intensity of NMS-873 treated cells. Drug treatment and timeline were the same as 3D. n=3 biological repeat. Data is mean ±S.E.M n.s.no significant from student’s t test. J Quantification of CFP fluorescent intensity of LLoMe treated cells. Drug treatment and timeline were the same as 3D. n=3 biological repeat. Data is mean ±S.E.M. n.s. no significant from student’s t test. K CFP fluorescent intensity of αS biosensors treated with scrambled or VCP siRNA. n=4 biological repeat. Data is mean ±S.E.M