Cell lysate protein concentrations were measured by bicinchoninic acid assay (Pierce, Rockford, IL) per the manufacturer’s instructions. Protein concentrations were measured with 10 µl of lysate and 200 µl of working reagent at absorbance of 570 nM with a microplate reader (Bio-Rad). All samples were analyzed in duplicates and absorbance values averaged. Concentrations were calculated by comparison to a bovine serum albumin standard curve.
An equal amount of lysate protein (ranging from 1–5 µg) was loaded onto Bis-Tris XT denaturing 10% SDS polyacrylamide gels (Bio-Rad). Proteins were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) at 200 V for 1.3 h and transferred onto polyvinylidene difluoride by electroblotting at either 30 V for 10 h or 100 V for 1.5 h. Blots were stained with Ponceau-S (Sigma) and visually inspected. Membranes were blocked for 1 h in 5% non-fat milk and incubated overnight with primary antibodies against APP (22C11, Chemicon, Billerica, MA), Dicer (NeuroMAB, Davis, CA), and α-tubulin (B-5-1-2, Sigma-Aldrich). Secondary antibody was HRP-conjugated goat anti-mouse (Rockland Immunochemical, Gilbertsville, PA) or HRP-conjugated rabbit anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Bands were visualized using ECL reagent (Pierce, Rockford, IL), detected on film and scanned for densitometric analysis.
An equal amount of lysate protein (ranging from 1–5 µg) was loaded onto Bis-Tris XT denaturing 10% SDS polyacrylamide gels (Bio-Rad). Proteins were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) at 200 V for 1.3 h and transferred onto polyvinylidene difluoride by electroblotting at either 30 V for 10 h or 100 V for 1.5 h. Blots were stained with Ponceau-S (Sigma) and visually inspected. Membranes were blocked for 1 h in 5% non-fat milk and incubated overnight with primary antibodies against APP (22C11, Chemicon, Billerica, MA), Dicer (NeuroMAB, Davis, CA), and α-tubulin (B-5-1-2, Sigma-Aldrich). Secondary antibody was HRP-conjugated goat anti-mouse (Rockland Immunochemical, Gilbertsville, PA) or HRP-conjugated rabbit anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Bands were visualized using ECL reagent (Pierce, Rockford, IL), detected on film and scanned for densitometric analysis.