Fig. 4: PBK phosphorylates and regulates multiple C2H2-ZF proteins in mitosis.
a Schematic of the Ikaros protein illustrating positions of zinc fingers (ZFs) 1–4, involved in DNA binding, and ZFs 5–6, involved in dimerisation. ZFs 1 and 2 (red) and the conserved linker sequence (black) are shown in more detail, along with the proposed PBK mitotic phosphorylation site16. An anti-phospho-linker antibody was generated against a phosphorylated peptide corresponding to the shaded sequence and used to probe mitotic lysates from Pbk+/+ or Pbk–/– mouse preB cells. b Western blot detection of proteins recognised by anti-phospho-linker antibody in asynchronous (Asyn.) and mitotically arrested (Mit.) Pbk+/+ and Pbk–/– mouse preB cell lysates. GAPDH was used as a loading control, and H3S10p was included as a mitotic marker. Representative of three independent experiments, with results further validated in a second PBK KO clone. Source data are provided as a Source Data file. c Heatmap representation of LC-MS/MS analysis of proteins immunoprecipitated with anti-phospho-linker antibody from Pbk+/+ mitotic lysates, compared to Pbk–/– control mitotic lysates (n = 3 + 3). Normalised LFQ intensity values, for three independent replicates, are shown for factors (111) which were detected after IP from Pbk+/+ mitotic lysates, but which were undetected or at significantly lower levels after IP from Pbk–/– lysates (modified two-tailed t test with permutation-based FDR < 0.05 and S0 = 0.1; see Methods for full filtering criteria). Proteins containing a C2H2-ZF domain are highlighted in red and constitute 72% of identified candidates. d Immunofluorescence staining of the indicated factors in fixed WT (Pbk+/+) or Pbk–/– mitotic mouse preB cells from asynchronously dividing cultures. Images are representative of > 16 mitotic cells from across two (YY1, CTCF) or three (SP1, Ikaros) independent staining experiments. Staining patterns were further validated in a second PBK KO clone (SP1, CTCF, Ikaros) and with an alternative antibody (SP1). Ikaros staining is derived from the experiment shown in Fig. 2d and a further representative image is included here for comparison. Scale bar = 5 µm. a Partially created in BioRender. Dimond, A. (2025) https://BioRender.com/x87bt2h.
