Fig 2: NPAS4 forms a complex with NuA4 across brain regions.a. Lysate from the hippocampus of KA-stimulated mice, fractionated by molecular weight (MW) using gel filtration and non-denaturing size exclusion chromatography. Western blotting for NPAS4 in the different MW fractions confirms that NPAS4 resides in a high MW complex peaking ~1 MDa in size. Representative image from 3 experiments. See gel source data (Supplementary Fig. 1). b. Sequence validation of Flag HA epitope tag appended to the C-terminus of Npas4 or Arnt2 in the P0 or F1 generation in Npas4-FH or Arnt2-FH mice. c. Left: Immunohistochemistry of NPAS4 and HA antibody staining in hippocampus of Npas4-FH mice, 6 h enriched environment. Scale bars: 100 µm. Right: Validation of Arnt2-FH knockin mouse line by immunohistochemistry in CA1. Scale bar 25 µm. Representative images from 3 experiments. d. Validation by Western blot that NPAS4 and NPAS4-FH have the same induction kinetics and specificity for membrane depolarization-induced calcium signaling. To stimulate cultured cortical neurons, 55 mM KCl was applied for the indicated time points. Representative image from one experiment. See gel source data (Supplementary Fig. 1). e. Confirmation by Western blot of NPAS4-FH expression and Flag immunoprecipitation (IP) in cultured cortical neurons from either wild-type or Npas4-FH mice. Representative image from one experiment. See gel source data (Supplementary Fig. 1). f. Confirmation via anti-Flag immunoprecipitation (IP) and Western blot that NPAS4-NuA4 also assembles in the visual cortex following 2 h light stimulation. Representative image from 2 experiments. See gel source data (Supplementary Fig. 1). g. Experimental diagram detailing glycerol gradient fractionation and immunoprecipitation of intact NPAS4-NuA4 complexes from high molecular weight (MW) fractions. h. Western blot of glycerol gradient fractions of NPAS4-FH in mouse cortical lysates segregated by MW. NPAS4 migrates in high MW fractions along with NuA4 components TRRAP, EP400, and DMAP1. Representative image from 3 experiments. See gel source data (Supplementary Fig. 1). i. Reciprocal validation of NPAS4-NuA4 interaction via Flag immunoprecipitation and Western blotting from high MW fractions of cortical lysates from wild-type, Npas4-Flag-HA (Npas4-FH), and Tip60-Flag (Tip60-F) mice. Representative image from 2 experiments. See gel source data (Supplementary Fig. 1). j. Reciprocal validation of NPAS4-NuA4 interaction via Flag immunoprecipitation and Western blotting from low MW fractions of cortical lysates from wild-type, Npas4-Flag-HA (Npas4-FH), and Tip60-Flag (Tip60-F) mice. Representative image from 2 experiments. See gel source data (Supplementary Fig. 1).