Estudo Funcional de Mutantes da Proteína Prion Celular Associados à Doenças por Prion

with STI1 rescued retinal and hippocampal neurons from pro-


Transfection of cell lines and isolation of PrP


-expressing cells

grammed cell death through activation of protein kinase A (PKA) (Chiarini et al., 2002; Zanata et al., 2002; Lopes et al., 2005). Additionally, PrP C–STI1 binding also induced the differentia- tion of hippocampal neurons by ERK1/2 activation (Lopes et al., 2005). Understanding the fate of both STI1 and PrP C after their association at the cell surface may help clarify the mechanisms associated with the neurotrophic roles of PrP C, and its possible bearing on loss-of-function components of prion diseases.


SN56 cells were plated on coverslips for 2 d and transfected using the

liposome-mediated method (Effectene; Qiagen) according to the manu- facturer’s instruction using a 1:10 ratio of DNA to Effectene. After6h of transfection, cells were differentiated in serum-free medium supple- mented with 1 mM dibutyryl-cAMP (Sigma) for 2 or 3 d with medium changes every day. In cotransfection experiments, a total of 3–5 j.Lg of DNA was used with a plasmid ratio of 1:4 of clathrin–GFP, flotillin1– GFP, and caveolin-1–GFP and a ratio of 1:2 for GFP-PrP C and dynamin I or…