Fig. 8
From: Autophagy regulates lipid metabolism through selective turnover of NCoR1
Specific interaction of NCoR1 with GABARAP. a Pull-down assay. One-Strep-FLAG (OSF)-tagged LC3B or GABARAP family proteins were expressed in HEK293T cells. Precipitates generated with Strep-Tactin Sepharose were subjected to immunoblot analysis with indicated antibodies. ULK1 and p62 bind to GABARAP family and all Atg8 family proteins, respectively, and were therefore used as positive controls. Data are representative of three independent experiments. b Diagrams of the deletion-mutation constructs of NCoR1 (left panel) and the corresponding pull-down assays (middle and right panels). Middle panel: Each FLAG-tagged NCoR1-deletion mutant and OSF-GABARAP was co-expressed in HEK293T cells. At 48 h post-transfection, lysates were prepared and precipitated with Strep-Tactin Sepharose. Right panel: OSF-GABARAP immobilized on Strep-Tactin Sepharose was mixed with lysates prepared from cells expressing each NCoR1-deletion mutant. The resultant precipitates were subjected to immunoblotting with anti-FLAG antibody. Data are representative of three independent experiments. c Alignment of the LC3-interacting region (LIR)/GABARAP-interacting motif (GIM) of NCoR1 homologs in various species. Black and gray boxes indicate identical amino-acid residues with complete and partial conservation, respectively. d Pull-down assay. OSF-GABARAP and FLAG-NCoR1 or NCoR1 ΔLIR/GIM were co-expressed in HEK293T cells. The assay was carried out as described in a
