Fig. 5: Phosphorylation of CDCA3 by CK2 promotes its degradation by the APC/CCdh1.
a Beeswarm plot showing CDCA3 levels assessed in control or CK2 depleted H460 cells that were treated with or without cisplatin. CDCA3 levels determined by analysis of high throughput immunofluorescence microscopy images. Data points represent CDCA3 levels within individual cells from a minimum of 1200 cells (see Supplementary Fig. 3a). Horizontal lines indicate median values (unpaired Student’s t test, ****P = < 0.0001). b Densitometry quantification of western blot analysis in Supplementary Fig. 3b, with dot points representing average log2 of relative endogenous CDCA3 levels from NSCLC cell lines treated with or without CX-4945. Representative of three independent experiments with lines connecting respective untreated and cisplatin treated cell lines (paired Student’s t test, *P = 0.0126). c Immunoprecipitation analysis of endogenous CDCA3 to assess the CK2-mediated phosphorylation of CDCA3 in untreated or cisplatin treated H460 cells. d Immunoprecipitation analysis of ectopic CDCA3 from H460 cell lysates to assess the impact of cisplatin, CX-4945 or combination of both upon CK2-mediated phosphorylation of CDCA3. e Immunoprecipitation analysis to assess CK2-mediated phosphorylation of enriched endogenous CDCA3 from nocodazole arrested H460 cells in prometaphase (prometa.) or H460 cells arrested at the G1/S cell cycle boundary by double thymidine block. c–e CK2 phosphorylation assessed using antibody specific for CK2 phosphorylation motifs within substrates. f Immunoprecipitation analysis of ectopic HA-Cdh1 with ectopic wild-type CDCA3-FLAG from H460 cells treated with cisplatin, CX-4945 or a combination of both. All in vitro experiments are representative of three independent repeats.
