Excelgel

Cells cultured at 1 g and in RPM condition for 24 and 48 hours were lysed in RIPA buffer (Sigma-Aldrich). Samples were then clarified by centrifugation at 10000 rpm for 10 min. Equivalent amount of protein (10 μg) from each sample was electrophoretically resolved on 12.5% precast SDS-polyacrylamide gels (ExcelGel, GE Healthcare Biosciences) using horizontal apparatus (Pharmacia Biotech, Uppsala, Sweden). Then, separated proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell) by a semidry system (Novablot, Pharmacia Biotech). Membranes were blocked with 3% nonfat milk in PBS and then were incubated (ON at 4°C) with the LC3B monoclonal antibody (1 : 2000; Sigma). After extensive washing with PBS containing 0.1% tween-20 (TBST), blots were incubated with 1 : 2000 dilution of HRP-conjugated secondary antibody (Amersham Biosciences) for 1 hour at RT. Immunopositive bands were detected with a chemiluminescence's detection system (GE Healthcare Biosciences). To check for equal loading of the gel, membranes were stripped and reprobed with mouse anti-β-actin antibody (1 : 20000, Sigma) and with anti-GAPDH antibody (1 : 1000, Cell Signalling Technology). Densitometric analysis was performed with the Quantity One software (BioRad Laboratories).

Treated and untreated cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2, Sigma). Samples were clarified by centrifugation at 1000 rpm for 5 min. Equivalent amount of protein (10 μg) from each sample was electrophoretically resolved on 12.5% precast SDS-polyacrylamide gels (ExcelGel, GE Healthcare Biosciences) using horizontal apparatus (Pharmacia Biotech, Uppsala, Sweden). Then, separated proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell) by a semidry system (Novablot, Pharmacia Biotech). Membranes were blocked with 5% BSA in PBS and then were incubated (overnight at 4°C) with the following monoclonal antibodies: GSK3α/β (Sigma), phospho-GSK3α/β (Ser 21/9) (cell signalling). After extensive washing with PBS containing 0.1% tween-20 (TBST), blots were incubated with 1 : 2000 dilution of HRP-conjugated secondary antibody (Amersham Biosciences) for 1 hour at room temperature. Immunopositive bands were detected with a chemiluminescence's detection system (GE Healthcare Biosciences). To check for equal loading of the gel, membranes were stripped and reprobed with mouse anti-β-actin antibody (1 : 20000, Sigma). Densitometric analysis was performed with the Quantity One software (BioRad Laboratories).

Samples of WT or D76N β₂m, 50 μm, were incubated in PBS, pH 7.4, at 37 °C under stirring in the presence of 50 μm D76N β₂m fibrils grown in the absence or presence of 10 μm α-crystallin. Aggregation of β₂m was monitored by quantifying the soluble fractions of WT and D76N β₂m by 8–18% polyacrylamide gradient gels (ExcelGel, GE Healthcare) and by ThT fluorescence emission at 480 nm after excitation at 445 nm (13 (link)).