Figure 2.

p85β is targeted for ubiquitylation and degradation by SCF^FBXL2. (a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated. (b) HeLa cells stably expressing FLAG–HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected. (c) During a 72-h serum starvation, NHFs were transfected with either an siRNA targeting FBXL2 (no. 1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (d) HEK293T cells were transfected with HA-tagged p85β, FLAG-tagged FBXL2, FLAG-tagged FBXL2(ΔF-box) or an empty vector (EV) as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of p85β was performed in the presence of E1, E2s and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (MeUb) was also added. Samples were analysed by immunoblotting with the indicated antibodies. The ladder of bands with a relative molecular mass of >85,000 (lane 3) corresponds to ubiquitylated p85β. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Uncropped images of blots are shown in Supplementary Fig. S8