Fig. 3
From: The IAP family member BRUCE regulates autophagosome–lysosome fusion
BRUCE interacts specifically with GABARAP and GABARAPL1. a, b Interaction of BRUCE wild type (WT) or aa 1–1648 fragment and mammalian ATG8 proteins and ubiquitins examined by GST pull-down assay. Total cell lysate of HEK293T cells transfected with Myc-tagged WT or the 1–1648 fragment was incubated with GST-ubiquitin or GST-ATG8 proteins immobilized with glutathione sepharose beads. Interaction was detected by immunoblotting using anti-Myc antibody. The amount of GST-protein used was compared by Coomassie or Ponceau S staining. c Interaction of BRUCE 1–1648 and GABARAP WT or mutants examined by GST pull-down assay. GABARAP mutants contained substitutions in the canonical LC3-interacting region (LIR) recognition surface (Y49A and Y49A/L50A). d, e Normalized mCherry/GFP signals under starvation condition normalized to FM condition in MEF lines stably expressing mCherry-EGFP-GABARAP d or mCherry-EGFP-GABARAPL1 e. Control (WT and shRenilla) and KD (shAtg5 and shBruce#1 and #2) cells were starved for 6 h. Data are presented as mean±SD from three biological replicates (****p < 0.0001, ***p < 0.001, NS, not significant). Representative data are shown from three independent experiments
