Fig. 1: Nuclear ANLN directly interacts with Pol II large subunit.

A Representative immunofluorescence staining images of ANLN (pink) and DNA (blue) in different cell cycles. Scale bar, 5 μm. B Western blotting was performed to examine the different subcellular fractions of the indicated cells. Lamin B1 and histone H3 were used as marker proteins of the chromatin. GAPDH was used as a marker of the cytoplasm. C BrUTP was transfected into HeLa cells using liposomes for 15 min, and then cultured in fresh medium for another 15 min. Immunofluorescence staining was performed using ANLN (green) and BrdU (red) antibodies. Scale bar, 5 μm. D The enrichment analysis of ANLN interacting proteins identified by mass spectrometry was performed using Metascape database. Based on the selected terms, accumulative hypergeometric P-values and enrichment factors were calculated and used for filtering. Remaining significant terms were hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. The 0.3 kappa score was applied as the threshold to cast the tree into term clusters. E Immunoprecipitation was performed using ANLN antibody, and the indicated antibodies were subsequently used for the western blotting. F The indicated plasmids were transfected into HEK293T cells for 48 h, and then the cells were lysed and subjected to immunoprecipitation with HA magnetic beads. G, H The interaction between recombinant POLR2A fragments and ANLN full-length protein was examined using an in vitro pulldown assay. I Representative PLA images for the interaction between endogenous ANLN and Pol II in KYSE510 cells. Scale bar, 5 µm. The data are representative of at least three independent experiments with similar results. Source data are provided as a Source Data file.