Figure 3.

De-NEDDylation decreases the half-life of L11. (A) H1299 cells were transfected as indicated. A measure of 30 μg/ml of cycloheximide (CHX) was added and cells were lysed in 2 × SDS sample buffer at the indicated time points. Equal amounts of protein were analysed by western blotting. The signals were quantified and presented as percentage difference in intensity. (B) Flag-L11, His₆-NEDD8 and β-gal constructs were transfected and an experiment with CHX was performed as above. Cells were collected and used for either Ni²⁺ pull-down or direct western blotting. Quantification of signals from five independent experiments was performed as above.