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. 2016 Mar 14;113(13):3527–3532. doi: 10.1073/pnas.1519389113

Fig. S4.

Fig. S4.

Additional intracellular experiments. (A) Interaction between FLAG-Ubv.Fw7.5 and exogenously expressed HA-Fbw7 or HA-Fbl1 in cell lysates. FLAG immunoprecipitates were separated on gel electrophoresis and probed for the presence of the indicated HA-tagged F-box proteins. Ubv.Fw7.5 coimmunoprecipitates significant levels of Fbw7, whereas Fbl1 presence in immunoprecipitates could not be detected by this assay. It should be noted that the levels of Fbw7-HA in cell lysates are significantly greater in the presence of Ubv.Fw7.5 expression; this is the result of inhibition of Fbw7 auto-ubiquitination (19) by the Ubv.Fw7.5. (B) Validation of Fbw7, Fbw1, and Fbw11 siRNA. Cells expressing FLAG-Fbw7 (Upper) and FLAG-Fbw1, FLAG-Fbw11 and FLAG-Fbw1+HA-Fbw11 (Lower) were treated either with control siRNA (“C”) or the siRNAs directed against the indicated proteins. (C and D) Expression of Ubv.Fw7.5 (C) and Ubv.Fw11.2 (D) dimer or monomer selectively stabilizes substrates of SCFFbw7 and SCFFbw11, respectively., but not other SCF ligases. Cells were transiently transfected with either siRNA molecules (positive control), empty vector (vector), or vectors expressing FLAG-Ubv. Cells were treated with CHX for the indicated time points and cell lysates were probed with endogeneous antibodies against the indicated proteins [substrate (F-box protein)]. (E) Expression of Ubv.Fw11.2 does not affect stability of Cdc25A and Wee1 in the background of Fbw11 siRNA treatment. (F and G) Distribution of G1-phase, G2-phase, and S-phase populations in cells expressing Ubv.Fw7.5 (F) and Ubv.Fw11.2 (G). Cells were transiently transfected with either siRNA molecules (positive control), empty vector (vector), or vectors expressing FLAG-Ubv. Analysis of cell cycle kinetics was determined by Hoechst dye (nucleic acid stain) staining, followed by flow cytometry analysis. The graph combines data from three biological replicates and mean ± SE is shown. (G) The effect of Fbw11 siRNA treatment and Ubv.Fw11.2 expression was assessed in the background of Fbw1 siRNA treatment.