Western Blotting of Zebrafish Proteins

Western blotting was performed as described previously (Ding et al., 2016 (link)). Embryos from 6 days postfertilization (dpf) or fresh hearts isolated from adult fish were transferred immediately to RIPA buffer (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail (Roche) and homogenized using a Bullet Blender tissue homogenizer (Next Advance). The resultant protein lysates were subjected to western blotting using a standard protocol. The following primary antibodies were used: anti-actin (1:8000, Santa Cruz Biotechnology, sc-1615), anti-phospho-mTOR (Ser2448) (1:2000, Cell Signaling Technology, 2971), anti-phospho-S6 ribosomal protein (Ser240/244) (1:5000, Cell Signaling Technology, 2215), anti-S6 ribosomal protein (1:8000, Cell Signaling Technology, 2217), anti-phospho-4E-BP1 (Thr37/46) (1:1000, Cell Signaling Technology, 2855), anti-4E-BP1 (1:2000, Cell Signaling Technology, 9644), anti-ubiquitin (1:1000, Thermo Fisher Scientific, PA5-17067) and anti-LC3 (1:3000, Novus Biologicals, NBP100-2331).