FIGURE 4.

Aurora-A is a substrate for USP2a in vitro and in vivo. A, recombinant His-tagged full-length Aurora-A was ubiquitinated with a ubiquitination kit as described under “Materials and Methods.” USP2a activity assays were carried out using the purified ubiquitinated Aurora-A with either buffer alone, 0.5 μg of USP2a, or 0.5 μg of USP2a-C276A as described under ”Materials and Methods.” The reaction mixture was subjected to Western blot analysis for ubiquitin, Aurora-A, and USP2a. B, H1299 cells were transfected with 1 μg of pcDNA3-Aurora-A and 1 μg of pcDNA3-His₈-ubiquitin alone, or in the presence of 1 μg of the following constructs: pcDNA3, pcDNA3-USP2a, pcDNA3-USP2a-C276A, or pcDNA3-Myc-USP7. Cells were treated with 30 μm MG-132 for 4 h. Cell lysates were prepared, and ubiquitinated proteins were purified using Ni-NTA beads and blotted with antibody for Aurora-A. Cell lysates were also blotted for Aurora-A, USP2a, and USP7 (bottom panel). C, cell lysates prepared in B were also blotted with anti-ubiquitin antibody.