Cells or embryos (at least 100 for conditions) were lysed in RIPA buffer containing 1X protease inhibitor cocktail (SIGMA). Cleared proteins extracts were quantified by using the Bradford method (Bio-Rad). For WB, protein samples were separated on 15% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked in TBS containing 5% Non Fat Dry Milk (Cell Signaling) and incubated with the follows primary antibodies: anti-GFP 1:500 (Abcam, ab13970), anti GFP rabbit polyclonal antibody (Life technologies, a6445), used 1:1000 (for HEK 293T/17 cells experiments), anti-β-Actin 1:500 (Sigma), anti-GAPDH 1:1000 (Santa Cruz Biotechnology), anti-COX-IV 1:500 (Cell Signaling), anti-COX7B 1:500 (Abcam, ab137094).
Proteins of interest were detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:3000, GE Healthcare) and rabbit anti-chicken IgY antibody (Millipore) visualized with the Luminata Crescendo substrate (Millipore) or the Super Signal West Femto substrate (Thermo Scientific), according to the manufacturer’s protocol. Western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using ImageJ software.
Proteins of interest were detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:3000, GE Healthcare) and rabbit anti-chicken IgY antibody (Millipore) visualized with the Luminata Crescendo substrate (Millipore) or the Super Signal West Femto substrate (Thermo Scientific), according to the manufacturer’s protocol. Western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using ImageJ software.