FIGURE 2.

Identification of HERC2 as a protein that interacts with FBXL5. A, HEK293T cells stably expressing FLAG-tagged FBXL5 were incubated for 6 h in the presence of MG132 (10 μm) and then subjected to immunoaffinity purification with agarose-conjugated antibodies to FLAG. The purified proteins were fractionated by SDS-PAGE and stained with silver. The positions of bands corresponding to various purified proteins are indicated. B, the proteins found to interact with FLAG-FBXL5 in A were identified by LC-MS/MS analysis. For clarity, only HERC2 and previously identified FBXL5-interacting proteins are listed. The peptide count as well as the percentage sequence coverage for each protein are also shown. C, HEK293T cells transfected (or not) with expression vectors for FLAG-SKP2, FLAG-tagged WT, or mutant (PE/AA) forms of FBXL5, FLAG-FBXW5, or FLAG-FBXW7α were subjected to immunoprecipitation (IP) with antibodies to FLAG, and the resulting precipitates as well as a portion of the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. D, HEK293T cells were incubated for 4 h in the presence of MG132 (5 μm) and then subjected to immunoprecipitation with antibodies to HERC2 (or with normal control IgG), and the resulting precipitates as well as a portion of the original cell lysates (Input) were subjected to immunoblot analysis with antibodies to FBXL5 and HERC2. The asterisk indicates a nonspecific band.