Figure 1: Identification of SCF^Fbw7 substrates.

(a) Screen design and proteomic workflow for global identification of candidate Fbw7 targets. Subcellular fractionation of HCT116 FBW7 WT and KO cells into cytosolic (Cyt) and nuclear-/organellar-(Nuc/org) enriched fractions was followed by TMT labelling, sample pooling and peptide fractionation by HiRIEF. Analysis of quantitative data by PQPQ⁴⁶-SAM identified upregulated proteins. (b) Venn diagram showing the total number and overlap of (1) the proteins found by quantitative proteomics to be upregulated in KO cells (2) proteins with ≥3 Fbw7 degron motifs (RKS/TPRKXS/T/E/D) and (3) proteins with motifs predicted to be phosphorylated by GSK3. (c) Validation of quantitative MS-data. WB analysis comparing endogenous protein levels in the different fractions from WT and KO cells. Equal protein amount from each cell type were analysed. (d) FBW7 KO cells were transiently transfected with Fbw7 and GSK-3β as indicated for 24 h. Whole cell extracts were analysed by WB.