Figure 6.

NEDD4 protein directly binds to N-Myc protein and targets N-Myc protein for ubiquitination. (a) BE(2)-C cells were transfected with control siRNA, NEDD4 siRNA or SIRT2 siRNA for 48 h. Cytoplasmic and nuclear protein was separated and analyzed by immunoblot with anti-NEDD4, anti-SIRT2, anti-GAPDH (marker for cytoplasmic protein), and anti-E2F1 (marker for nuclear protein) antibodies. (b) HEK293 cells were transfected with constructs expressing empty vector, N-Myc and/or NEDD4. Protein from the cells was immunoprecipitated with an anti-N-Myc Ab or a control mouse Ab, and IPproducts were analyzed by immunoblot with an anti-NEDD4 Ab. (c) HEK293 cells were transfected with constructs expressing HA-ubiquitin, Flag-N-Myc and/or Flag-NEDD4. Upper panel: protein from the cells was immunoprecipitated with an anti-N-Myc Ab, and co-IP products were analyzed by immunoblot with an anti-HA Ab. Lower panel: 5% of whole-cell lysates was used as input and probed with an anti-Flag Ab. (d) HEK293 cells were transfected with constructs expressing Flag-N-Myc and Flag-NEDD4. Protein from the cells was precipitated with anti-Flag M2 beads and incubated with E1, a panel of E2 enzymes, HA-ubiquitin (HA-Ub), and ATP. Polyubiquitinated protein was detected by immunoblot (IB) with an anti-HA Ab (upper panel). The indicated smear appearing above 110 kDa represented polyubiquitinated NEDD4 and N-Myc (N-Myc-UBⁿ), and the smear/ladder between 76 and 110 kDa represented N-Myc-UBⁿ. Immunoblot results with an anti-Flag M2 Ab showing Flag-tagged products are shown in the lower panel. (e) HEK293 cells were transfected with constructs expressing Flag-N-Myc, Flag-NEDD4, mutant Flag-NEDD4, or empty vector. Protein from the cells was precipitated with anti-Flag M2 beads, and precipitated protein was incubated with E1, UbcH5a, HA-ubiquitin, and ATP, followed by immunoblot analysis with an anti-HA Ab