FIG 1.

NRF1 interacts with the OGT/HCF-1 complex. (A) Silver staining of NRF1 nuclear complex. Nuclear extracts of 293F cells expressing NRF1-3×FLAG and those with an empty vector (mock) were pulled down with an anti-FLAG antibody. (B) Detection of OGT and HCF-1 proteins in NRF1 nuclear complex shown in panel A by immunoblot analysis. (C) Constructs of 3×FLAG fusion proteins of NRF1 deletion mutants. 3×FLAG-NRF1 WT (1–741), 3×FLAG-NRF1 ΔbZip (1–592), 3×FLAG-NRF1 M1 (Δ464–580), and 3×FLAG-NRF1 M2 (Δ243-580) were expressed in 293F cells and immunoprecipitated. (D) Detection of OGT and HCF-1 proteins interacting with NRF1 and its mutant molecules. Nuclear extracts of 293F cells expressing NRF1 and its mutant molecules were pulled down with an anti-FLAG antibody. Immunoprecipitated samples were subjected to immunoblot analysis with antibodies against OGT, HCF-1, and the FLAG tag. (E) Constructs of His₆-tagged proteins of NRF1 mutants and GST fusion proteins of OGT and the C-terminal half of HCF-1 (HCF-1-C). Also shown are NRF1-Neh5L/AD2-His₆ (Fr. 1; 243–430) and NRF1-Neh6L-His₆ (Fr. 2; 431–580). (F) Coomassie brilliant blue staining of GST fusion proteins and His₆-tag proteins. GST-OGT, GST–HCF-1-C, NRF1-Neh5L/AD2-His₆ (Fr. 1; 243–430), and NRF1-Neh6L-His₆ (Fr. 2; 431–580) were bacterially expressed and purified. Arrowheads indicate purified recombinant fusion proteins. (G) GST pulldown assay of NRF1-His₆ fragments using GST-OGT and GST–HCF-1-C. NRF1 fragments were detected using an anti-His₆ antibody, and OGT and HCF-1 were detected using an anti-GST antibody.