Figure 8.

Ubiquitination, but not NEDDylation, is required for the rapid degradation of SCRAP-GFP. (A) EL4/SCRAP-GFP cells were treated with 2.5 μM Shield-1 overnight to allow accumulation of SCRAP-GFP. The cells were then washed, resuspended in fresh media, supplemented with either 10 μM MG132, 10 μM MLN7243, 10 μM MLN4924, or an equivalent volume of DMSO (mock), and incubated at 37 °C. GFP fluorescence was detected in each of the samples via flow cytometry every 1.5 h over 6 h and the MFI of the population reported. (B) Cell lysates of EL4 cells stably expressing NC NEDD8-GFP following a 3-h treatment with 10 μM MLN7243, 50 μM 3MA, and 10 μM MLN4924 were analyzed by Western blotting for GFP or p97. All Western blot images were cropped to show relevant bands.