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

 

PKC activity


incubated with 100 f,LM IBMX for 1 h at 37°C, followed by either Ln `)`1 peptide (37 f,LM), at different time points (0.5 to 10 min), or with forskolin (10 f,LM) for 20 min at 37°C as a positive control. Cells were washed with cold PBS and homog- enized in ice-cold extraction buffer (150 mM NaCl, 20 mM MgCl2, 1% Triton X-100, and 25 mM Tris-HCl, pH 7.4) plus Complete Protease Inhibitor Cocktail (Roche). Cellular  de- bris was removed by centrifugation at 6000 g for 10 min. PKA activity was determined by [32P] `)`-ATP incorporation to a PKA-specific substrate provided by the PKA assay system kit (Upstate Biotechnology). The reaction was done according to the  manufacturer’s  instructions.

 

ERK1/2 activity

 

Primary hippocampal (1X106 cells) neurons from Prnp+/+ or Prnp0/0 mice were plated on dishes pretreated with poly-l- lysine and stimulated with Ln `)`1-chain peptide (37 f,LM) at various time points (0.5 to 30 min). Cells were also treated for

0.5 min with Ln `)`1 peptide (37 f,LM) after a preincubation either with or without U0126 (10 f,LM), KT5720 (60 nM), BIM

(0.5 f,LM), and U73122 (1 f,LM) for 30 min. Cells were rinsed with ice-cold PBS and lysed in Laemmli buffer. Cell extracts were subjected to SDS-PAGE (10%), and proteins were trans- ferred onto nitrocellulose membranes. The membranes were…