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. 2020 Apr 29;6(18):eaaz8031. doi: 10.1126/sciadv.aaz8031

Fig. 2. Direct interaction between the inhibitor and COUP-TFII protein.

Fig. 2

(A) CETSA was performed using LNCaP cells. COUP-TFII overexpressed 293T cells (B) or LNCaP cells (C) were used for biotinylated inhibitor pulldown assay. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation. Twenty micromolar CIA1 was used as competitor. (D) Biotinylated inhibitor pulldown assay using COUP-TFII fragments overexpressed 293T cells. Flag-CII-C (C-terminal 147 to 414 amino acids) and Flag-CII-N (N-terminal 1 to 182 amino acids). (E) Biotinylated inhibitor pulldown assay using purified glutathione S-transferase–tagged COUP-TFII ligand-binding domain (GST-LBD; 172 to 414 amino acids). GST antibody was used for Western blot assay. (F) Biotinylated inhibitor pulldown assay using overexpressed COUP-TFII with amino acid mutation in 293T cells. WT, wild type. (G) 293T cells that transfected with NGFIA-Luc and mutated COUP-TFII were treated with CIA1 for 18 hours. Luciferase assay was performed. n = 3 per group. (H) Biotinylated inhibitor pulldown assay using overexpressed nuclear receptors in 293T cells. Twenty micromolar CIA1 was used as competitor. HA, hemagglutinin.