Fig. 9

Functional characterization of NPAS3 variants. a Western blots showing that NPAS3 variants are normally expressed when transfected into HEK 293T cells. b HaloTag pull-down data demonstrating that the p.Val304Ile and p.Ala552Pro variants are able to interact with ARNT. Bands in the top panel of pull-down lane of ‘prey’ protein, indicating physical association of HA-NPAS3 variants with the HaloTag-ARNT construct. Middle and bottom panels are results for the HaloTag-clone and HaloTag-empty constructs. As HaloTag constructs are covalently linked to the resin, they will be depleted in the pull-down sample, demonstrating functionality of the HaloTag. Molecular weight of HaloTag-ARNT, 121 kDa, HaloTag construct with no cDNA: 34 kDa, Full-length NPAS3: 101 kDa. c, d Luciferase reporter expression demonstrating transactivation function of all three variants tested. The previously characterized variant p.Val304Ile was tested in two isoforms in (c). The remaining variants, p.Ala552Pro and p.Gly697Ser was tested in isoform 1. Reporter used is luciferase (luc2) expression driven by a construct including 797 bp proximal to the transcription start site of VGF. RLU = relative luciferase units, the ratio of firefly luciferase and renilla luciferase to normalize for transfection efficiency. Top and bottom of each box indicate the 25th and 75th centile, internal bar indicates the median, whiskers indicate data extremes. Data are representative of three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001