Fig. 1

NONO directly interacts with nuclear PKM2. (A) Proteins biotinylated by BioID2 alone or NONO-BioID2 in MDA-MB-231 cells were examined via western blotting with HRP-conjugated streptavidin following SDS‒PAGE separation. PKM2 was identified via quantitative mass spectrometry and indicated based on its molecular weight. (B) Endogenous PKM2 was coimmunoprecipitated with anti-Flag M2 Sepharose beads in MDA-MB-231 and BT-549 cells overexpressing Flag-tagged NONO. IgG served as the negative control. (C) Coimmunoprecipitation of endogenous NONO with PKM2-Flag from MDA-MB-231 and BT-549 cells. (D) The cellular localization of NONO and PKM2 in MDA-MB-231 cells was examined by immunofluorescence with anti-NONO and anti-PKM2 antibodies and by nuclear counterstaining with DAPI. (E) A GST pull-down assay was used to detect the interaction between NONO and PKM2 in vitro (top). Purified GST and GST-NONO fusion proteins preabsorbed to glutathione-Sepharose beads were incubated with the prokaryotic His-PKM2 fusion protein. GST and GST-NONO fusion proteins were visualized by Coomassie blue staining (bottom). (F) Schematic diagram of NONO truncation mutants (top). The binding of His-PKM2 fusion proteins to purified GST, GST-NONO fragments F1 (1-143 aa), F2 (1-228 aa), F3 (144–471 aa), and F4 (229–471 aa) was assessed by a GST pull-down assay (middle). GST, GST-NONO F1, F2, F3, and F4 fusion proteins were visualized by Coomassie blue staining (bottom). (G) Schematic diagram of PKM2 in different truncated constructs (top). The binding of the His-NONO fusion protein to purified GST, GST-PKM2 fragments F1 (1-116 aa), F2 (117–218 aa), F3 (219–389 aa), and F4 (390–531 aa) was assessed by a GST pull-down assay (middle). GST, GST-PKM2 F1, F2, F3, and F4 fusion proteins were visualized by Coomassie blue staining (bottom)