Supplementary Figure 2: Identification of the KBP degron.

(a–d) HEK293T cells were transfected with either an empty vector (EV), wild type human KBP, or the indicated FLAG-tagged human KBP mutants (either deletion, truncation, or point mutation mutants, as indicated). Whole cell extracts (WCE) were immunoprecipitated (IP) with an anti-FLAG resin, and immunoprecipitates were probed with the indicated antibodies. (e) Alignment of the Fbxo15 binding motif in KBP orthologs. The critical lysine required for Fbxo15 binding is highlighted in red. (f) Schematic representation of KBP mutants. KBP mutants found to interact with Fbxo15 are indicated by the symbol (+). (±) denotes reduced binding, and (−) denotes a lack of binding. (g) HEK293T cells were transfected with either an empty vector (EV), wild type human KBP, or the indicated FLAG-tagged human KBP point mutation mutants. Whole cell extracts (WCE) were immunoprecipitated (IP) with an anti-FLAG resin, and immunoprecipitates were probed with the indicated antibodies. (h) Strep-FLAG-tagged human KBP was immunoprecipitated from HEK293T cells and subjected to PTM analysis. The table lists the number of spectra matching acetylated and phosphorylated peptides as a fraction of the total spectra detected for peptides bearing each modified residue and their corresponding modification percentage. This experiment was performed once. (i) HEK293T cells were transfected with either an empty vector (EV) or vectors expressing FLAG-tagged wild type human KBP or FLAG-tagged human KBP(KK/RR). Whole cell extracts were immunoprecipitated (IP) with anti-FLAG resin, and proteins were immunoblotted as indicated. Unprocessed original scans of blots are shown Supplementary Fig. 9. Unless otherwise noted, experiments were performed at least three times.