Fbxw4 interacts with E3 ubiquitin ligase components and with the COP9 signalosome in an F-box dependent manner.

<p>A. Schematic of the Fbxw4^−fbox protein. Numbers represent the amino acids of the protein. Domains contained within Fbxw4^−fbox are indicated. B. Table representing the number of unique peptides identified from one representative mass spectrometry experiment following FLAG immunoprecipitation from lysates of control cells expressing FLAG only “contr.”, or cells expressing FLAG- Fbxw4 or FLAG- Fbxw4^−fbox. Column on left indicates the size of the interacting protein, in kilo-daltons (kDa). Gene names of proteins that contain the identified peptides are shown in the right column. Components of an E3 ubiquitin ligase complex are shaded in light gray; components of the COP9 signalosome are shaded dark gray. C. Validation of data mass spectrometry data by immunoprecipitation followed by western blot. 293 T cells were transfected with plasmids containing FLAG-Fbxw4, FLAG-Fbxw4^−fbox, FLAG-Fbxo46 or an empty vector (v). 48 hours post-transfection 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 SKP1.</p

The Fbxw4 locus is a common site of proviral insertion and is highly expressed in the murine mammary gland during involution.

<p>A. Examination of the UCSC genome browser (genome.ucsc.edu) and the Retroviral Tagged Cancer Gene Database (<a href="http://variation.osu.edu/rtcgd/index.html" target="_blank">http://variation.osu.edu/rtcgd/index.html</a>) shows that multiple retroviral insertions have been cloned from within the transcribed Fbxw4 locus. Arrows indicate the directionality of the inserted provirus. The exons are indicated at the bottom and the position and scale of murine chromosome 19 is shown on top. Names given to the cloned proviral insertions are indicated on the left. Insertion sites beginning with ‘mmt’ were cloned from mouse mammary tumor virus induced mammary carcinomas. Insertion sites beginning with ‘Dkm’, ‘248’ and ‘B5’ were cloned from leukemias from murine leukemia virus accelerated hematopoietic cancers. B. FBXW4 is variably expressed in normal murine tissues. Oligos specific for murine Fbxw4 were used to perform quantitative rt-PCR on the ‘mouse normal cDNA TissueScan array’. Values were normalized to quantitative rt-PCR for GAPDH. C. Schematic of the Fbxw4 protein. Numbers represent the amino acids of the protein. Domains contained within Fbxw4 are indicated.</p

Fbxw4 interacts with components of an E3 ubiquitin ligase complex and the COP9 signalosome.

<p>A. Table representing the number of unique peptides identified from one representative mass spectrometry experiment following FLAG immunoprecipitation from lysates of control cells expressing FLAG only “contr.”, or cells expressing FLAG-Fbxw4 or FLAG-Fbxo46. Column on left indicates the size of the interacting protein, in kilo-daltons (kDa). Gene names of proteins that contain the identified peptides are shown in the right column. Components of an E3 ubiquitin ligase complex are shaded in light gray; components of the COP9 signalosome are shaded dark gray. B. Validation of data mass spectrometry data by immunoprecipitation followed by western blot. 293 T cells were transfected with plasmids containing FLAG- Fbxw4, FLAG-Fbxo46 or an empty vector (v). 48 hours post-transfection cell lysates were prepared and immunoprecipitations were performed with mono-clonal anti-FLAG antibodies (M2) (to immunoprecipitate Fbxw4- or Fbxo46-interacting complexes). Western blots were performed to detect Fbxw4 or Fbxo46 (FLAG rb; polyclonal FLAG antibody; top panel), SKP1, COPS5, or COPS2. C. Expression of Fbxw4 alters the migration of endogenous SKP1 by gel filtration chromatography. 293 T cells were transfected with an empty vector (left panels) or a cplasmid containing FLAG-Fbxw4 (right panels). 48 hours post-transfection cell lysates were prepared and separated on a superpose6 gel filtration column. Western blots were performed on every other fraction to detect Fbxw4 (top panels) or SKP1 (bottom panels). In the absence of Fbxw4 SKP1 elutes with a peak at fraction 23, whereas when Fbxw4 is expressed there is co-elution of Fbxw4 with peaks at fraction 15 and in the void volume. Size standards that elute from given fractions are shown.</p

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.</p