Filter Trap Assay

The filter trap assay was performed as described (Vos et al., 2010). In brief, bafilomycin A-treated cells were counted and a fixed number were lysed in 200μl FTA buffer (10mM Tris-HCl, pH8.0, 150mM NaCl) containing 2% SDS. Samples were boiled for 10 minutes and loaded onto a pre-washed (FTA, 0.1% SDS) 0.2μm cellulose acetate filter (GE Healthcare) with supporting filter paper (Bio-Rad) in a Bio-Dot 96 well filtration system (Bio-Rad). Suction was applied to filter the samples. Following loading, membranes were processed as described above in Western Blotting.