Fig. 4: NUPR1 physically interacts with ESR1.

A Immuno-purification of NUPR1-containing protein complexes. Cellular extracts from MCF-7TamR cells stably expressing FLAG (empty vector, control) or FLAG-NUPR1 were immunopurified with an M2 anti-FLAG affinity gel and eluted with 3× FLAG peptide. The eluates were resolved by SDS-PAGE, and bands of interest were analyzed by mass spectrometry. *, nonspecific binding proteins. B DuoLink assay of the interaction between FLAG-tagged NUPR1 and endogenous ESR1 (red) in MCF-7TamR cells treated with vehicle (ethanol) or 0.6 μM Tam for 24 h. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. C Representative immunofluorescence (IF) images of endogenous NUPR1 and ESR1 colocalization in MCF-7TamR but not in their parental cells. Green, ESR1; Red, NUPR1. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. D, E Flag-tagged NUPR1 or ESR1 was transfected into MCF-7TamR cells with or without Tam treatment for 48 h. Immunoprecipitation (IP) of whole-cell lysates with an M2 anti-FLAG antibody and Western blotting analysis of the indicated proteins were conducted. IgG h. c. IgG heavy chain, IgG l.c. IgG light chain. F Co-IP assay of NUPR1 and ESR1. FLAG-tagged ESR1 or its serial deletions was transfected into MCF-7TamR cells and Western blotting analysis of the indicated proteins were conducted as in D, with ACTB as a loading control. Left: schematic representation of full-length ESR1 protein and its truncated forms. A/B, activating function; C, DNA-binding domain; D, nuclear localization signal; E and F, hormone-binding domain.