G9917809

The cells and tissue samples were homogenized in lysis buffer (50 mM pH 7.5 HCl-Tris, 300 mM NaCl, 0.5% SDS, and 1% Triton X-100) and incubated 15 min at 95°C. Protein concentration of the extracts was measured using the DC protein assay kit (500–0111, Bio-Rad). Equal amounts of total protein extract from healthy and SAD cells were resolved by SDS-PAGE and then transferred to nitrocellulose (G9917809, Amersham, Germany) or PVDF (IPVH00010, Merk Millipore, Cork, Ireland) membranes. Western blot and immunoreactive proteins were developed using an enhanced chemiluminescence detection kit (NEL105001EA, Perkin Elmer) following instructions of the supplier. Quantification was performed by densitometry of the obtained bands in each lane with respect to the correspondent housekeeping protein such as GAPDH or β-tubulin band in each experiment (Quantity One software, Bio-Rad).

The cells and tissue samples were homogenized in lysis buffer (50 mM pH 7.5 HCl-Tris, 300 mM NaCl, 0.5% SDS and 1% Triton X-100) and incubated 15 min at 95°C. Protein concentration of the extracts was measured using the Dc protein assay kit (500-0111, Bio-Rad). Equal amounts of total protein extract from control and FAD cells were resolved by SDS-PAGE and then transferred to nitrocellulose (G9917809, Amersham, Germany) or PVDF (IPVH00010, Merk Millipore, Cork, Ireland) membranes. Western blot and immunoreactive proteins were developed using an enhanced chemiluminescence detection kit (NEL105001EA, Perkin Elmer) following instructions of the supplier. Quantification was performed by densitometry of the obtained bands in each lane with respect to the correspondent housekeeping protein in each experiment (Quantity One software, Bio-Rad). When molecular weights were different enough, sometimes the same membrane was re-used to detect several proteins. In this way, the housekeeping protein pattern might appear in different western blots for different proteins. When indicated, data was normalized with respect to the values obtained from each correspondent age-matched control sample.