Fig 2: 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

Fig 3: Immunodetection of binding partners of PRDM14. A, Eluents of a Halo pull‐down assay were analyzed by Wes using anti‐PRDM14, anti‐HSP90α, anti‐GRP78, anti‐γ‐Catenin, and anti‐caspase‐14 antibodies. B, C, Co‐immunoprecipitation (Co‐IP) experiments were carried out with lysates prepared from Halo‐PRDM14‐transduced HCC1937 and MDA‐MB231 cells. The lysates were immunoprecipitated with anti‐HSP90α, anti‐GRP78, or anti‐γ‐Catenin antibodies and then immunoblotted (IB) with (B) each antibody and (C) anti‐Halo antibody. GRP78, glucose‐regulated protein 78; HSP, heat shock protein

Fig 4: Halo pull‐down assay followed by mass spectrometry. Halo pull‐down assay was carried out using Halo‐PRDM14‐transduced MDA‐MB231 and HCC1937 cells. Pull‐down samples were analyzed by SDS‐PAGE detected by silver stain. The 4 bands in the enlarged view (arrowheads) were analyzed by mass spectrometry