Western Blot Analysis of Spastin Proteins

COS-7 cells and zebrafish embryos were respectively lysed in SDS-lysis buffer [25 mM sodium phosphate (pH 7.2), 5 mM EDTA, 1% SDS] and SDS sample buffer [0.5 µl per embryo; 1 M Tris-HCl (pH 6.8)/10% glycerol/5% β-mercaptoethanol/3.5% SDS], supplemented with a cocktail of protease inhibitors (Roche). A total of 10 µg of zebrafish protein extracts and 5 µg of total protein lysates from COS-7 mock and transfected cells were electrophoresed into 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. Immunoblotting was performed after overnight incubation at 4°C with HA (1:5000, 11867423001, Roche), spastin86-340 (1:1000; Connell et al., 2009 (link)), H2b (1:16,000, ab1790, Abcam) and actin (1:10,000, AC-40, Sigma) antibodies. Immunostained proteins were visualised using appropriate peroxydase-labelled antibodies (Jackson ImmunoResearch) and a chemiluminescence detection system (Santa Cruz Biotechnology). DrM1 and DrM61 levels were estimated by quantifying blot band density normalised to H2b values (ImageJ software).