Pfn31k expression vectors

HEK293 cells (ATCC) were cultured in DMEM (Gibco) + 10% FBS (Seradigm) with incubation in a humidified 37 °C/5% CO₂ incubator. N- or C-terminal NLuc/PLK fusions were encoded in pFN31K expression vectors (Promega), including flexible Gly-Ser-Ser-Gly linkers between NLuc and ORFs corresponding to UniProt isoform 1 (PLK1: P53350-1, PLK2: Q9NYY3-1, and PLK3: Q9H4B4-1). Carrier DNA was encoded in pGEM-3Z vectors (Promega). For cellular BRET target engagement experiments, HEK293 cells were transfected with NLuc/PLK fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, NLuc/PLK fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:9 (mass/mass), after which FuGENE HD was added at a ratio of 1:3 (µg DNA: µL FuGENE HD). One part (vol) of FuGENE HD complexes thus formed was combined with 20 parts (vol) of HEK293 cells suspended at a density of 2 × 10⁵ per mL, followed by incubation in a humidified 37 °C/5% CO₂ incubator for 20 hr.

HEK293 cells (ATCC) were cultured in DMEM (Gibco) + 10% FBS (Seradigm), with incubation in a humidified, 37 °C/5% CO₂ incubator. N-terminal NLuc-PLK1, PLK2 or PLK3 fusions were encoded in pFN31K expression vectors (Promega). Carrier DNA was encoded in pFN5K vectors (Promega). For cellular BRET target engagement experiments, HEK-293 were transfected with NLuc/target fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, NLuc/target fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:9 (mass/mass), after which FuGENE HD was added at a ratio of 1:3 μg DNA: μL FuGENE HD). 1 part (vol) of FuGENE HD complexes thus formed were combined with 20 parts (vol) of HEK-293 cells suspended at a density of 2 × 10⁵ per mL, followed by incubation in a humidified, 37 °C/5% CO₂ incubator for 20 hr.