Zebrafish Protein Extraction and Western Blotting

Following our previous protocol (Ding et al., 2011 (link)) with modification, larval zebrafish proteins were extracted with lysis buffer and separated using 12% SDS-polyacrylamide gel electrophoresis. The protein bands were transferred onto a nitrocellulose membrane and blocked the membrane with TBS containing 5% skim milk. The membranes were incubated either with mouse anti-LC3 antibody (M186-3; MBL Biotechnology, Inc.) at 1:1,000 dilution or with rabbit anti-P62 antibody (PM 045; MBL Biotechnology, Inc.) at 1:2,000 dilution in TBS containing 1% skim milk; then the membrane was washed and incubated with secondary antibodies (HRP-conjugated goat anti-mouse or goat anti-rabbit IgGs; both 1:2,000 dilutions; Zhongshanjinqiao Co. China) for 2 h at RT. Chemiluminescent signals were detected using the Supersignal H West Pico chemiluminescent substrate (Thermo) with a GE Imaging System (GE Corporation).