Quantifying Mitochondrial and Cytoplasmic Hemoglobin

Western blotting was conducted as previously described (Shephard et al., 2014 (link)). Primary antibodies used were: Hba sc-21005 (Santa Cruz) 1:1000; and ab102758 (Abcam) 1:500 – for fly hypoxia; Hbb sc-22718 (Santa Cruz) 1:1000; COXIV ab16056 (Abcam) 1:1000; beta-actin ab8227 (Abcam) 1:4000; HSP-90 ab13495 (Abcam) 1:500; VDAC/Porin ab15895 (Abcam) 1:2000; NDUFS3 ab110246 (Abcam) 1:1000; SMAC/Diablo ab8115 (Abcam) 1:1000; dilution in 3% (w/v) BSA in TBS-T. Band densities were measured using Image J and samples were normalised to beta-actin. Using the normalised values the ratio of mitochondrial/cytoplasmic HbA and HbB were calculated. Data were analysed using the R statistical package http://www.r-project.org/ see supplemental data for script used to generate Fig. 3.