Fig. 3: Identification of the proteolytic cleavage site in the DR1 domain of Plk3.
a Top, the Plk3 protein disorder plot was generated by GeneSilico MetaDisorder server (http://genesilico.pl/metadisorder/). The Solvent accessibility was predicted by RaptorX server. Bottom, the Plk3 domain diagram matching the disorder tendency is shown. b Schematic diagram of Flag-tagged Plk3 and mutants with DR1 region and the NRDC cleavage site depicted. c–e Colony-formation assay (c, d) and flow cytometry analysis of apoptosis-inducing activity (e) from PANC-1 cells transfected with indicated Flag-tagged Plk3 or mutants (Plk3NT1-353 is p41Plk3). f Immunoblots of p72Plk3 and p41Plk3 (left) and cleaved PARP (right) at the indicated times in PATC50 cells transfected with Flag-tagged WT p72Plk3 or Plk3 mutants and grown under suspension conditions. g, h Immunoblot of p72Plk3 and p41Plk3 expression (g) and flow cytometry analysis of apoptosis-inducing activity (h) from PDAC cells transfected with indicated Flag-tagged Plk3-WT or mutants and grown under suspension conditions for 36 h. i Immunoblot of p72Plk3 and p41Plk3 expression in PATC148 cells that were lentivirally transduced to express the sgRNA targeting NRDC or non-targeting control sgRNA, and then stably transfected with p72Plk3 or p72Plk3R354G under a Dox-inducible system. j Immunoblot of p68Plk1 and p38Plk1 expression in PANC-1 cells that were stably transfected with Plk1 or Plk1R337G after the CRISPR/Cas9 deletion of NRDC. Cells in i and j were grown in suspension culture for 48 h. k PATC148 cells stably expressing Dox-inducible p72Plk3 or p72Plk3R354G were lentivirally transduced to express NRDC WT or H233G/H237G/E236G mutant. Lysates were subjected to immunoblot to assess the cleavage of Plk3. Data are representative of two independent experiments with similar results (f, g, i–k). Error bars, mean ± SEM (d, e, h), n = 3 independent experiments, two-tailed unpaired t-test (d, e, h). Source data are provided as a Source Data file.
