Figure 1

Fbw7-mediated ubiquitylation and stability control of human c-Myb protein. (A) Interaction between human c-Myb and human Fbw7. HEK293 cells were transfected with Myc-tagged human c-Myb in the absence or presence of HA-tagged human Fbw7. Cell lysates were subjected to immunoprecipitation (IP) with antibodies against HA, and the resulting precipitates as well as the original cell lysates (input) were subjected to immunoblotting (IB) with antibodies against Myc or Fbw7. (B) HEK293 cells were transfected with expression constructs for Myc-tagged human c-Myb with or without FLAG-tagged human Fbw7 and HA-tagged ubiquitin (Ub) as indicated. The cells were then incubated with MG132. To precisely demonstrate the polyubiquitylation of c-Myb, cell lysates were subjected to double IP with antibodies against Myc or mouse IgG. The resulting precipitates as well as the original cell lysates (input) and the first IP precipitates were subjected to IB with anti-HA, anti-c-Myb or anti-FLAG antibodies. (C) HeLa cells were transfected with human c-Myb in the absence or presence of HA-human Fbw7. For the cycloheximide (CHX) assay, cells were prepared after the indicated times of chase incubation and subjected to IB. The percentages of c-Myb remaining after the various chase times were quantified by image analysis. The immunoblots shown are representative of four independent experiments.