Ab18738

All protein (zebrafish embryos and mouse tissue) was extracted in RIPA buffer (ThermoScientific) with 1% protease inhibitor cocktail (Sigma) and homogenised. Protein concentration was determined by BCA assay (ThermoScientific). SDS-page was performed using 20% pre-cast NuPage 4–12% BisTris gradient gels (Life Technologies) and transferred to PVDF membranes by the iBlot 7 minute semi-dry blotting system (Life Technologies). Following blocking, membranes were incubated overnight at 4°C using mouse anti-SMN (1:1,000, BD biosciences, 610153), rabbit anti-PGK1 (1:1000, Millipore, ABS787), rabbit anti-ATP5A (1:1000 ab176569, Abcam), rabbit anti-cyt C (1:1000, Abcam, ab18738) mouse anti-COX IV (1:1000 Abcam, ab14744), mouse anti- GAPDH (1:1000, Abcam, ab9484,). After PBS washes, secondary antibodies were added for 1 hour at room temperature. Secondary antibodies used were; IR dye 800CW goat anti-mouse IgG (926–32210), IR dye 800CW goat anti-rabbit IgG (926–3211), IR dye 680RD goat anti-mouse IgG (926–68070), IR dye 680RD goat anti-rabbit IgG (926–68071), all 1:5,000, LI-COR Biosciences. Membranes were imaged using an odyssey infrared imaging system (Li-COR, Biosciences) as described previously [80 (link)], and quantified using an image studio software (Li-COR). To determine final relative protein expression, band intensities were normalized to a loading control or a ponceau stain.