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Estudo Funcional de Mutantes da Proteína Prion Celular Associados à Doenças por Prion
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Biotinylation of cell-surface proteins was performed as described pre- viously (Ribeiro et al., 2005; Lee et al., 2007). Briefly, cells were incubated with STI1 or 500 j.LM Cu 2+ for 5 or 10 min, respectively, transferred to ice, washed, and incubated on ice in PBS/CM (PBS supplemented with
1.0 mM MgCl2 and 0.1 mM CaCl2). Cell-surface proteins were biotinyl- ated with 1 mg/ml sulfo-NHS-SS-biotin (Pierce) for 1 h on ice. To
software or MetaMorph. The total fluorescence inside cells after a 40 min
incubation with STI1-AF568 was quantified. Images were thresholded, and the total fluorescence was detected automatically and independently by the software. The results were expressed as the mean of total fluores- cence per cell. For colocalization indices, cells were analyzed using Meta- Morph, by independently counting fluorescent objects (vesicles) and analyzing the percentage of colocalization independently by the software.
Conditioned medium from astrocytes
Primary mouse astrocyte cultures were obtained as described previously
(Lima et al., 2007). After reaching confluence, cells were maintained in DMEM without serum for 48 h. The conditioned medium (CM) was collected, centrifuged for 10 min to remove cellular debris, and filtered in
0.2 j.Lm membranes. CM (total volume of 30 ml) was concentrated to a
final volume of 150 j.Ll (200X) in Minicon Static concentrator B12…
1.0 mM MgCl2 and 0.1 mM CaCl2). Cell-surface proteins were biotinyl- ated with 1 mg/ml sulfo-NHS-SS-biotin (Pierce) for 1 h on ice. To
software or MetaMorph. The total fluorescence inside cells after a 40 min
incubation with STI1-AF568 was quantified. Images were thresholded, and the total fluorescence was detected automatically and independently by the software. The results were expressed as the mean of total fluores- cence per cell. For colocalization indices, cells were analyzed using Meta- Morph, by independently counting fluorescent objects (vesicles) and analyzing the percentage of colocalization independently by the software.
Conditioned medium from astrocytes
Primary mouse astrocyte cultures were obtained as described previously
(Lima et al., 2007). After reaching confluence, cells were maintained in DMEM without serum for 48 h. The conditioned medium (CM) was collected, centrifuged for 10 min to remove cellular debris, and filtered in
0.2 j.Lm membranes. CM (total volume of 30 ml) was concentrated to a
final volume of 150 j.Ll (200X) in Minicon Static concentrator B12…
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