FBXW4 associates with ubiquitinated cellular proteins.

<p>A. Fbxw4 can be immunoprecipitated by ubiquitinated proteins and Fbxw4 can immunoprecipitate ubiquitinated proteins. 293 T cells were transfected with empty vector (v), HA-Ubiquitin, or HA-Ubiquitin and FLAG-Fbxw4. 36 hours post-transfection cells were treated with MG132 (+) or left untreated (−) for six hours. Cell lysates were prepared and immunoprecipitations were performed with either anti-HA antibodies (top two panels) or mono-clonal anti-FLAG antibodies (M2) (bottom two panels). Western blots were performed on both sets of immunoprecipitations with anti-FLAG and anti-HA antibodies. B. FBXW4 associates with cellular proteins that are endogenously ubiquitinated. 293 T cells were transfected with plasmids containing FLAG-Fbxw4, FLAG- Fbxw4^−fbox, FLAG-Fbxo46 or an empty vector (v). 36 hours post-transfection cells were treated with MG132 (+) or left untreated (−) for six hours. Cell lysates were prepared and immunoprecipitations were performed with mono-clonal anti-FLAG antibodies (M2) (to immunoprecipitate Fbxw4-, Fbxw4^−fbox-, or FBXO46-interacting complexes). Western blots were performed to detect Fbxw4, Fbxw4^−fbox or Fbxo46 (FLAG rb; polyclonal FLAG antibody; top panel) or Ubiquitin.