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

 

Protein detection

Fluorescent-labeled proteins (STI1 and STI1<l230 –245;2 j.Lg) were submit- ted to 10% SDS-PAGE. To visualize labeled proteins, the gel was irradi-

ated with ultraviolet light, and images were acquired using a UV transil- luminator MultiDoc-It-Digital Imaging System (Bioimaging Systems). Alternatively, proteins resolved in 10% SDS-PAGE were subjected to immunoblotting with polyclonal antibody anti-STI1 (1:10,000) (Zanata et al., 2002). Rabbit nonimmune-purified IgG was used as the immuno- blotting negative control. For detection of PrP C, cell extracts were pre- pared by homogenizing the cell pellet in lysis buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% acid deoxycholic) and a protease inhibitor mixture (complete protease inhib- itor tablets; Roche Diagnostics) or a protease inhibitor mixture (Sigma) at twice the concentration suggested by the manufacturers. Proteins from cells were resolved in SDS-PAGE, transferred to nitrocellulose or Immobilon-P transfer membranes (Millipore), and incubated with mouse anti-PrP C antibody (Zanata et al., 2002). Staining was revealed by enhanced chemiluminescence (ECL Plus; GE Healthcare) or Super Sig- nal Chemiluminescent Substrate (Pierce).

 

Fluorescence imaging

Confocal microscopy was performed using a Bio-Rad MRC 1024 laser-

scanning confocal system running the Lasersharp 3.0 software coupled to a Zeiss microscope (Axiovert 100) with a 100 X 1.4 numerical aperture (NA) or 63 X 1.3 NA oil-immersion lens (Zeiss), a Bio-Rad Radiance 2100 laser-scanning confocal system coupled to a…