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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Biochim Biophys Acta Mol Cell Res. 2020 Jun 26;1867(10):118786. doi: 10.1016/j.bbamcr.2020.118786

Figure 1. HTS pipeline and results for discerning mechanisms of Nav channel complex regulation by kinases.

Figure 1.

(A) Screening and validation pipeline. (B) Double stable HEK293 cells expressing CLuc-FGF14 and CD4-Nav1.6-NLuc were plated in 384-well plates and treated with kinase inhibitors (n = 1 compound/well) from the Broad, Selleck, and UTKinase collections, with each plate screened in triplicate. The mean percent luminescence (normalized to on-plate 0.3% DMSO controls) is shown for each compound. Following exclusion of toxic compounds (purple), hits were initially selected using unbiased criteria of change in FGF14:Nav1.6 complex assembly by at least 40% (i.e., % luminescence > 140% or < 60%) and Z-score ≥ 3 (enhancers, green) or Z-score ≤ −4 (inhibitors, red). (C) Z’-Factor (Z’) for each screened library plate, calculated using either the inhibitor (red) or enhancer (blue) positive controls as described previously[25]. A total of 33 plates were screened, including 6 from Broad, 12 from Selleck, and 15 from UTKinase, for a total of 3,120 compounds.