Fig. 4

Identification of PNSC928 and determination of its in vitro and in vivo effects on disruption of CtBP2-p300 interaction. (A) A schematic representation illustrating four regions of the p300 amino acid sequence. (B) Co-IP results. Different combinations of plasmids were cotransfected into MPAEpiC cells. After a 48-hour incubation, cells were lysed and used for immunoprecipitation with anti-Flag agarose. The input and output proteins were probed with anti-Flag and anti-Myc antibodies. n = 3. (C) A schematic representation illustrating the in vitro AlphaScreen setup. (D) AlphaScreen signals for different concentrations of His-CtBP2 and GST-p300^BRD. (E) Chemical structure of PNSC928. (F) IC₅₀ of PNSC928 disrupting CtBP2-p300^BRD interaction. (G and H) In vitro pulldown assay in a reaction consisting of His-CtBP2, GST-p300^BRD, and incremental concentrations of PNSC928 (0, 11.8, 23.6, and 47.2 µM). (G) Pulldown with Ni-NTA beads; (H) Pulldown with GST beads. (I) In vivo immunoprecipitation with anti-CtBP2-coated protein G beads in MPAEpiC cells exposed to varying doses of PNSC928 (0, 11.8, 23.6, and 47.2 µM) over a 6-hour period. n = 3