A GLUC-based sensor to monitor autophagy. (a) Rapamycin (Rap) induces release of GLUC activity from Actin-LC3-dN. Actin-LC3-dN was transfected in 293ET cells and medium was replaced after 24 h with serum-free medium containing 200 nM rapamycin for 6 h before analysis of GLUC activity in SN. (b) ATG4B but not ATG4A induces cleavage of Actin-LC3-dN. SN of 293ET cells transiently co-transfected with Actin-dN or Actin-LC3-dN and GFP, ATG4A or ATG4B were collected after 24 h and analyzed for GLUC activity. Error bars were calculated from three independent transfections. RLU, relative light units. (c) ATG4B cleaves GFP-LC3. 293ET cells transfected with GFP-LC3 and ATG4A or ATG4B were lysed in 1% NP40, resolved by 10% SDS-PAGE and blotted with anti-GFP. Full-length GFP-LC3 (LC I) runs at 45 kDa and the cleaved product runs at 43 kDa (LC II). (d) ATG4B cleaves Actin-LC3-dN to generate a small LC3-dNGLUC fragment. 293ET cells transfected with Actin-LC3-dN and GFP or ATG4B were treated for 6 h with 10 μg/ml Brefeldin A (right panel) to block secretion of cleaved dNGLUC or left untreated (left panel) before lysis in 1% NP40. Whole cell lysates were resolved by 10% SDS PAGE and blotted with an antibody raised against dNGLUC. The protein band corresponding to full-length Actin-LC3-dN is marked with an asterisk, the cleavage product is marked with an arrowhead.
Ketteler et al. Genome Biology 2008 9:R64 doi:10.1186/gb-2008-9-4-r64