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Novel and adapted molecular strategies to create and trace toxic proteins in neurodegenerative disease Aron S. Workman, Hilda Slunt-Brown, and David R. Borchelt Department of Neuroscience and the McKnight Brain Institute University of Florida, Gainesville, FL 32612 I. PCR based mutagenesis II. Pulse chase coupled with nonionic detergent extraction III. Inducible neural cells IV Detecting superoxide radical antioxidants by riboflavin nitro blue tetrazolium assay 6 12 18 24 30 36 42 48 WT D 101G D 101N III. Inducible neural cells Analytical Biochemistry colored formazan superoxide antioxidant Light No dox 1 g/ mL dox HEK293 pTet On N2a pR Tet On 34 +contrast mouse neuroblastoma (line N2a) human neuroblastoma (line SH SY5Y) Retinoic acid differentiation aworkman@ufl.edu www.aronw.com S1 P2 35Scysteine pulse chase NP 40 insoluble protein aggregates soluble proteins and oligomers et al. Nature Success minipreps Fail minipreps MATERIALS AND METHODS Cell culture and transfections. SOD 1 aggregate extraction and immunoblotting g S1 P2 35Scysteine pulse chase analysis. Superoxide anion scavenging activity assay 1x TEN with 0.5% NP -40, 0.25% SDS, 0.5% deoxycholate and 1% PI et al Analytical Biochemistry