Figure 4.

Non-cleavable NEDD8-GFP undergoes autophagosomal degradation. (A) Western blot analysis of EL4/NC NEDD8-GFP cells following a 5-h treatment with autophagy inhibitors 3-MA or bafilomcyin. Cell lysates of NC EL4/NEDD8-GFP cells and NC EL4/NEDD8-GFP treated with inhibitors were made and analyzed by Western blot using mouse monoclonal antibodies against GFP and actin with appropriate secondary antibodies. (B) The average fold increase in GFP following autophagy inhibition was determined by Western blot analysis is reported for three independent experiments. (C) Flow cytometric analysis for GFP fluorescence of cells after autophagosomal inhibition are compared with mock-treated cells. (D) Western blot analysis of EL4/NC NEDD8-GFP cells after overnight treatment with 5mM NH4Cl treatment. (E) Western blot analysis of EL4/NC NEDD8-GFP cells transfected with either scrambled siRNA oligomers or siRNA targeting ATG7. Cell lysates were probed for either ATG7 (top panel) or p97 as a loading control (bottom panel). (F) EL4/NC NEDD8-GFP cells were transfected with scrambled or ATG7-targeting siRNAs and cultured with or without 3-MA. Cell lysates were examined by Western blot analysis for GFP. (G) GFP signal obtained by Western blot was quantified in three independent experiments. The increase of NC/NEDD8-GFP for each treatment is shown as the fold-increase increases over mock-treated control. Error bars represent the standard error. All Western blot images were cropped to show relevant bands.