Fig. 4.

Biological activity of Ubvs in HEK293T cells. (A) Ubv interaction partners identified by mass spectrometry of FLAG-Ubv immunoprecipitates from cell lysates. Spectral counts refers to number of peptides corresponding to each identified protein. Only proteins relevant to SCF ligases are shown (see Table S3 for complete list of detected proteins). (B) Expression of Fw7.5 Ubv in monomer or dimer disrupts interaction of Fbw7 with Cul1. HA-Fbw7 immunoprecipitates were probed for FLAG-Cul1 and endogenous Skp1, in the absence or presence of FLAG-Ubv expression. (C) Expression of Ubv.Fw11.2 Ubv in dimer format, but not monomer format, disrupts interaction between Fbw11 and Cul1. Analysis performed as described in B. (D and E) Expression of Ubv.Fw7.5 (D) and Ubv.Fw11.2 (E) in monomer or dimer format stabilizes the SCF^Fbw7 (Cyclin E and c-Myc) and SCF^Fbw11 (Cdc25A and Wee1) substrates, respectively. Cells were transiently transfected with either siRNA molecules (positive control), empty vector (Vector), or vectors expressing FLAG-Ubv. Cells were treated with cycloheximide (CHX) for the indicated time points and cell lysates were probed with antibodies against the indicated proteins. Quantification of relative substrate levels was performed using ImageJ and represents average of two independent experiments (see D and Fig. S4C for c-Myc and Cyclin E, and E and Fig. S4D for Cdc25A and Wee1). (E) The effect of Fbw11 siRNA treatment and Ubv.Fw11.2 expression was assessed in the background of Fbw1 siRNA treatment.