Split-luciferase complementation assay

Cells were trypsinized (0.25%), triturated in a medium, and seeded in white, clear-bottom CELLSTAR μClear^® 96-well tissue culture plates (Greiner Bio-One) at ∼10⁵ cells per well in 200 μL of medium.

For transiently transfected cells, trypsinization was performed 48 h post-transfection. The cells were incubated for 24 h, and the growth medium was subsequently replaced with 100 μL of serum-free, phenol red–free DMEM/F12 medium (Invitrogen) containing protein kinase inhibitors (1–50 μM) or TNF-α (1–50 ng/mL). The final concentration of DMSO was maintained at 0.3% or 0.5% for all wells excluding the positive control wells containing medium alone. Following 2 h incubation at 37 C, the reporter reaction was initiated by injection of 100 μL substrate solution containing 1.5 mg/mL of D-luciferin dissolved in PBS (final concentration = 0.75 mg/mL) by the Synergy™ H4 Multi-Mode Microplate Reader (BioTek, Winooski, VT). Luminescence readings were performed at 2-min intervals for 20–30 min, integration time 0.5 s, and the cells were maintained at 37 °C throughout the measurements. Signal intensity for each well was calculated as a mean value of peak luminescence; the calculated values were expressed as percentage of mean signal intensity in the control samples from the same experimental plate. For our assay, an enhancer is defined as a positive modulator and inhibitor is defined as a negative modulator of FGF14:Nav1.6 complex assembly, as detected through increases or decreases in luminescence, respectively.

Cells were trypsinized (0.25%), triturated in a medium, and seeded in white, clear-bottom CELLSTAR μClear® 384-well tissue culture plates (Greiner Bio-One) at 30 000 (3 × 10⁴) cells per well in 40 μL of serum-free, phenol red–free DMEM/F12 medium using the Multidrop Combi (Thermo Fisher). Nanoliter volumes of library compounds or controls (DMSO, TNF-α, MNS) are transferred from low dead volume source plates using the LabCyte Echo 550 acoustic transfer platform. The final concentration of DMSO was maintained at 0.3% for all wells excluding those wells containing cells with medium alone. Following 2 h incubation at 37 C, the reporter reaction was initiated by injection of 40 μL substrate solution containing 1.5 mg/mL of D-luciferin (final concentration = 0.75 mg/mL) by the Multidrop Combi. After 1 h incubation, luminescence intensity is read using a Tecan Infinite M1000.

D-luciferin was purchased from BioGold and prepared as a 30 mg/mL stock solution in phosphate- buffered saline (PBS). Recombinant human TNF-α was purchased from Abcam and dissolved in PBS containing 0.1 mg/mL BSA (for protein stability) and prepared as a 10 µg/mL stock solution. Triciribine and MNS were purchased from Tocris, and ZL181 was developed by the laboratory of Jia Zhou at UTMB; each drug was dissolved in dimethyl sulfoxide (DMSO) as 20 mM stock solutions. Drugs for the HTS were provided as 10 mM stock solutions in DMSO on 384-well plates.