Figure 3

Human Fbw7 mediates degradation of c-Myb via binding and ubiquitylation, but each system is independent and do not require the functional CPD. (A) Interaction between human or mouse c-Myb and human or mouse Fbw7. HEK293 cells were transfected with Myc-tagged c-Myb in the absence or presence of Fbw7. Cell lysates were subjected to immunoprecipitation (IP) with antibodies against HA or FLAG, and the resulting precipitates as well as the original cell lysates (input) were subjected to immunoblotting (IB) with antibodies against Myc or Fbw7 which signal corresponded to arrowhead. Asterisk shows non-specific signal. (B) Human or mouse Fbw7-dependent ubiquitylation of mouse and human c-Myb. For analyses of c-Myb ubiquitylation, HA-ubiquitin and Myc-tagged c-Myb in the absence or presence of human or mouse Fbw7 were transfected into HEK293 cells. The transfected cells were then incubated with MG132. Cell lysates were subjected to IB with an anti c-Myb to detect ubiquitylation of the c-Myb protein. Anti Fbw7 antibody was used to detect Fbw7, which signal corresponded to arrowhead. Asterisk shows non-specific signal. (C) Interaction between human c-Myb and human WT or arginine mutant Fbw7. HEK293 cells were transfected with human c-Myb in the absence or presence of FLAG-tagged human Fbw7. Cell lysates were subjected to IP with antibodies against FLAG, and the resulting precipitates as well as the original cell lysates (input) were subjected to IB with antibodies against c-Myb or FLAG. (D) Human Fbw7-dependent but arginine-independent ubiquitylation of human c-Myb. For analyses of human c-Myb ubiquitylation, HA-ubiquitin and human c-Myb in the absence or presence of human WT or arginine mutant Fbw7 were transfected into HEK293 cells. The transfected cells were then incubated with MG132. Cell lysates were subjected to IB with an anti c-Myb or anti FLAG to detect ubiquitylation of human c-Myb protein or Fbw7, respectively.