Fig. 1. Identification of proteins interacting with Apg5 in mouse ES cells. (A) Wild-type ES cells stably expressing GFP alone and Apg5-deficient ES cells stably expressing GFP-fused Apg5 (GFP24) were labeled with [³⁵S]methionine/cysteine for 2 hours. Following immunoprecipitation with anti-GFP antibody from cell lysates, the immunoprecipitates were analyzed by SDS-PAGE and a bioimage analyzer. (B) Purification of Apg5-interacting proteins. Total cell lysates were prepared from GFP24 cells and subjected to affinity purification using an anti-GFP antibody-coupled protein-A/Sepharose bead column. Following elution, bound proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The positions of unconjugated GFP-Apg5, Apg12/GFP-Apg5, and three unknown proteins of 63 kDa, 71 kDa and 144 kDa are indicated. Asterisk indicates the position of immunoglobulin heavy chain partially dissociated from the column.