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. 2024 Mar 21;15:2531. doi: 10.1038/s41467-024-46857-w

Fig. 2. GAS41 interacts NRF2 in vitro and in vivo.

Fig. 2

a Schematic representation of N-terminal Flag-HA-tagged NRF2 (FH-NRF2) plasmid (upper panel) and process of NRF2-interacted proteome identification (lower panel). b Coomassie blue staining of purified NRF2-associated protein from nuclear extraction from overexpressed FH-NRF2 H1299 stable cell line. The specific protein bands were cut and analyzed by mass spectrometry. c Western blot of GAS41-interacted protein complex of overexpressed SFB (S protein tag, Flag tag, and Streptavidin binding peptide)-GAS41 stable HEK293T cell line by two steps immunoprecipitations: first step IP by streptavidin beads and second step IP by S protein beads. d Western blot of interaction between overexpressed HA-GAS41 with Flag-NRF2 in HEK293T cells. e Immunoprecipitation of A549 cells by GAS41 antibody or control IgG was analyzed with western blot. f Immunoprecipitation of A549 cells by NRF2 antibody or control IgG was analyzed with western blot. g Schematic diagram of the GAS41 domains and GAS41 mutants used in this study. GAS41-amino acids 1–160 contains YEATS domain and A box (conserved sequence elements in YEATS proteins), referred to AA. 1–160; GAS41-amino acids 1–183 contains YEATS domain and A box, referred to AA. 1–183; GAS41-amino acids 1–206 contains YEATS domain, A box and partial coiled-coil motif, referred to AA. 1–206; GAS41-amino acids 161–227 contains complete coiled-coil motif, referred to AA. 161 to 227. h Western blot analysis of interaction between NRF2 and GAS41 mutants AA. 1–160 or AA. 161–227 in HEK293T cells. i Western blot analysis of interaction between NRF2 and GAS41 mutants AA. 1–160, AA. 1–183, or AA. 1–206 in HEK293T cells. Western blot experiments above (b–f, h–i) were repeated three times with similar results and representative images are shown. Source data are provided as a Source Data file.