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. 2009 Dec 11;11(1):45–51. doi: 10.1038/embor.2009.256

Figure 1.

Figure 1

Nix interacts with Atg8/LC3/GABARAP proteins. (A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD-W/-L/-H medium with a β-gal assay. (B) GST pulldown assays using cell extracts of Flag-Nix-expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies. (C) Purified GST-tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST-fusion proteins. (D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β-gal, β-galactosidase; BafA1, bafilomycin-A1; CCCP, carbonyl cyanide m-chlorophenyl hidrazone; GST, glutathione-S-transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin-like modifier 1; TCL, total cell lysate; TM, transmembrane domain.