Horseradish peroxidase conjugated anti mouse or anti rabbit secondary antibodies

Cells were lysed in NP‐40 buffer, and proteins were resolved by SDS–PAGE. After transferring the proteins onto PVDF membranes, HIF proteins were detected using anti‐HIF‐1α (mouse monoclonal, BD Bioscience 610958), anti‐HIF‐2α (mouse monoclonal, 190b) or anti‐HIF‐1β (rabbit polyclonal, Novus Biologicals, NB100‐110) antibodies and horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibodies (Dako). HRP‐conjugated anti‐β‐actin antibody (Abcam) was used as a loading control.

Fish were culled and collected, dried from excess embryo medium, and homogenised on ice with lysis buffer containing 1% octyl-glucoside, complete protease inhibitor cocktail and PhosSTOP tablets (Sigma, Burlington, MA, USA). The tissue was homogenized by sonication and cleared by centrifugation at 7000 rpm for 5 min at 4 °C. Supernatants were diluted in 2× Laemmli Buffer at 1:1 dilution, resolved by SDS-PAGE (10% and 12% gels) and transferred to PVDF membranes. The membranes were incubated in in 5% non-fat dry milk/PBST blocking solution for 1 h at room temperature and then the relevant primary antibodies were diluted in PBST overnight at 4 °C. Membranes were washed in PBST, incubated with 1:5000 dilution of horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Dako, Santa Clara, CA, USA) in PBST for 1 h at room temperature and then washed again in PBST. Bands were visualised using ECL™ (GE Healthcare Bioscience, Piscataway, NJ, USA). The amount of protein was normalized to tubulin and quantified using ImageJ (Fiji) software. The following antibodies were used: mouse anti-Tubulin (1:1000; Sigma-Aldrich), rabbit anti-Dendra (1:1000; Online Antibodies), mouse anti- α-Synuclein (1:1000; Insight Biotechnology, Wembley, UK), mouse anti-pSer129 (1:500; Biolegend, San Diego, CA, USA).