Figure 4.

NanoLuc luciferase‐based bioluminescence resonance energy transfer (NanoBRET) analysis in living cells. A, Schematic of NanoBRET assay for detecting intracellular protein‐protein interactions by measuring energy transfer between a NanoLuc (NLuc) donor fusion protein and a HaloTag acceptor fusion protein. NLuc with substrate excites fluorescence‐conjugated HaloTag ligand with HaloTag fusion protein by energy transfer. B, 293T cells were transfected with Halo‐PRDM14 and NLuc fusion proteins. Expression level of Halo‐PRDM14 was analyzed using Wes with anti‐PRDM14. GAPDH was used as a loading control. Expression levels of NLuc fusion proteins expressed in 293T cells were detected by NLuc substrate. C, D, A simplified NanoBRET donor saturation assay was carried out. 1:1, 1:10, 1:100, and 1:1000 dilutions of NLuc relative to HaloTag DNA were used. HSP90α‐NLuc and NLuc‐GRP78 were co‐transfected with (C) Halo‐PRDM14 and (D) Halo‐PRDM14‐ΔC measuring energy transfer. Error bars represent the mean ± SD of triplicate samples. GRP78, glucose‐regulated protein 78; HSP, heat shock protein