Filter trap assay

SDS-insoluble aggregates were analyzed by filter trap analysis as previously described (Nivon et al., 2012 ). Briefly, cells were scraped in 2% SDS-FTA buffer (FTA: 150 mM NaCl, 50 mM dithiothreitol, 10 mM Tris-HCl, pH 8). Next samples were homogenized by three passages through a 25-gauge needle. The 2.5-μg protein extracts were diluted by a factor of 2–8 (1, 1:2, 1:4, and 1:8) and applied with mild suction into a slot blot apparatus onto a 0.22-μm Protran BA83 nitrocellulose membrane (Schleicher and Schuell, Dutscher, Brumath, France) prewashed with 0.1% SDS-FTA buffer. Then the membrane was washed with 0.1% SDS-FTA buffer and 0.1% Tween-TBS buffer (TBS: 20 mM Tris-HCl, pH 7.6, 137 mM NaCl) and processed for immunoblotting. Equivalent expression levels of wt and mutant constructs for HspB5 and SOD1 were checked on Western blots of total cell lysates in parallel experiments.