Fig 2: KIF26B enhances nuclear localization of ZC3H13/CBLL1. A-B.CBLL1 and ZC3H13 mRNA expression in MDA-MB-157 (A) and NMFH-1 (B) cells with lentiviral mediated KIF26B knockdown. C–F. KIF26B, ZC3H13, and CBLL1 protein expression in the cytoplasmic (C), and nuclear (N) fractions from MDA-MB-157 (panel C and E) and NMFH-1 (panel D and F) cells with KIF26B knockdown alone (C-D) or in combination with WT-KIF26B overexpression (E-F). G. Immunofluorescence analysis of CBLL1 (green), ZC3H13 (green), and DAPI (blue, cell nuclei) in KIF26B knockdown and control MDA-MB-157 and NMFH-1 cells. Scale bar, 20 μm. H–I. METTL3, METTL14, VIRILIZER, and WTAP protein expression in cytoplasmic (C), and nuclear (N) fractions from MDA-MB-157 (H) and NMFH-1 (I) cells with or without KIF26B knockdown. J-K. Interactions between WTAP and METTL3, METLL14, VIRILIZER, CBLL1, and ZC3H13 were determined by co-IP using cytoplasmic fractions from MDA-MB-157 (J) and NMFH-1 (K) cells with or without KIF26B knockdown. n.s., not significant. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig 3: Semi-quantitation of nuclear/cytoplasma protein ratio from western blotting in Fig. 2. A-D. Semi-quantitation of nuclear/cytoplasma (N/C) CBLL1 and ZC3H13 ratio matched to each group in Fig. 2C (A), Fig. 2D (B), Fig. 2E (C), and Fig. 2F (D). E-F. Semi-quantitation of N/C METTL3, METTL13, VIRILIZER, and WTAP ratio matched to each group in Fig. 2H (E) and Fig. 2I (F). ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig 4: KIF26B interacts with ZC3H13 and CBLL1 in the cytoplasmic fraction of multiple cancer cell lines. A. IF staining of KIF26 (red), CBLL1 (green), and ZC3H13 (green) in MDA-MB-157, NMFH-1 and TT cells. B. Left panel, Co-IP was conducted using the cytoplasmic fraction MDA-MB-157 (top), NMFH-1 (middle), and TT (bottom) cells. Right panel, reciprocal IP assay was performed to explore the interaction of ZC3H13 and CBLL1 with KIF26B. KIF26B was immunoprecipitated by anti-CBLL1 and anti-C3H13. C and D. The structure of the wild-type full length (FL) and mutation constructs of Flag-tag labeled ZC3H13 (C) and CBLL1 (D). E and F. Flag-tagged FL or truncated mutation constructs were co-transfected with HA-KIF26B into NMFH-1 cells. The KIF26B/Flag-tag complexes were immunoprecipitated by anti-HA antibodies. Flag-ZC3H13 (E) and Flag-CBLL1 (F) proteins were detected using anti-Flag antibodies.