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Ere1 associates with an internalized plasma membrane protein, Can1, and colocalizes with Vps5. (A) ESCRT-mutant (vps23Δ) cells coexpressing an integrated Ere1-13xmyc fusion and Can1-3xHA (from its own promoter on a centromeric plasmid) were treated with cycloheximide for the indicated times and then cross-linked with DSP and DTBP. Can1-3xHA was immunoprecipitated with antibodies specific for HA from solubilized cell lysates. Immunoprecipitates were subsequently analyzed by Western blotting using antibodies against the myc or HA epitopes to detect Ere1 and Can1, respectively. (B) Ere1 and Vps5 colocalize on endosomal compartments. ESCRT-mutant (vps23Δ) cells coexpressing Ere1-RFPmars and Vps5-GFP (top) or coexpressing Ere1-RFPmars and Snx41-GFP (bottom) were examined by confocal microscopy.