Assay of α-SNAP release and displacement of Vps41 and Arl8

Fusion reactions of twice the standard volume were incubated under conditions specified in figure legends (Figure 1, Supplemental Figure S3, Figure 7, and Supplemental Figure S4). Reactions were stopped by addition of 1 ml of cold HB. Phagosomes and lysosomes were sedimented by centrifugation (16,000 × g, 30 min, 4°C), resuspended in 20 µl 1× SDS sample buffer, and analyzed for LAMP1 and α-SNAP or for LAMP1, Vps41, Arl8, and Rab7 by SDS–PAGE and immunoblotting. Immunoblots were quantified using ImageJ image processing software.