Anti cathepsin b

Protein lysates were prepared from P3 Grn^+/+ and Grn^−/− microglia using RIPA lysis buffer (1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 20 mM Tris pH 7.6, 150 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄) supplemented with protease inhibitor cocktail. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membranes were blocked in 5% non-fat dry milk (Bio-Rad) for 30 min before incubating with primary antibodies at 4°C overnight. Primary antibodies included anti-ADAM33 (Thermo Fisher, PA5-28128, 1:1000), anti-ATG7 (Abcam, ab133528, 1:3000), anti-Cathepsin B (Proteintech, 12216-1-AP, 1:2000), and anti-GAPDH (Millipore, MAB374, 1:4000), anti-MEF2C (Cell Signaling, 5030, 1:1000), anti-Myosin Va (Cell Signaling, 3402, 1:2000) and anti-Numb (gift from Dr. Yuh Nung Jan, 1:5000)⁴¹ . On the second day, the membranes were washed with 0.1% TBST washing buffer followed by incubation with secondary antibodies conjugated with HRP at room temperature for 1 hour. Secondary antibodies used for western blots included goat anti-mouse IgG (H+L), peroxidase conjugated (Millipore, 401215, 1:5000), and goat anti-rabbit IgG (H+L), peroxidase conjugated (Millipore, 401315, 1:5000).