Mouse anti gapdh

About 50 µg protein per lane were electrophoresed through an ExcelGel SDS gradient 8–18% polyacrylamide gel (GE Healthcare Life Sciences, Chicago) and blotted onto a nitrocellulose membrane. The membrane was incubated with anti-HBS1 antibody (1:500) or, for comparison, with mouse anti-GAPDH (HyTest, 1:5,000) at 4 °C overnight. Then the membrane was incubated with sheep anti-mouse immunoglobulin (GE Healthcare Life Sciences, 1:10,000) coupled to horseradish peroxidase for 1 h at room temperature, and bands were visualized using the enhanced chemiluminescence system (SuperSignal West Dura Extended Duration Substrate, Thermo Fisher Scientific).

Brains were lysed in a protein extraction buffer containing 50 mM Tris (pH 7.4), 2% SDS, and complete protease inhibitor cocktail (Roche, Mannheim, Germany) and homogenized by sonication. The insoluble debris was removed by centrifugation, and the protein concentration of the supernatant was measured by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Western blot analysis was performed as described previously [20 (link)]. Twenty microgram protein was loaded per lane on SDS polyacrylamide gels (ExcelGel SDS, gradient 8–18, Amersham Biosciences) on a horizontal electrophoresis system (Amersham Biosciences) and thereafter blotted onto a nitrocellulose membrane. For the detection of specific antigens, the following first step antibodies were used: rabbit polyclonal anti-p62 (BML-PW9860-0100, Enzo Life Sciences, NY, dilution 1:2000), rabbit anti-Atg7 (Sigma, 1:1000), and mouse anti-GAPDH (HyTest Ltd., Finland, 1:2000). As secondary antibodies, goat anti-rabbit immunoglobulin G (IgG) (Bio-Rad Laboratories, CA) and sheep anti-mouse immunoglobulin G (GE Healthcare, UK) antibodies conjugated to horseradish peroxidase were used at a dilution of 1:10000. The bands were revealed with enhanced chemiluminescence reagent (ThermoFisher Scientific).