Figure 5

Emodin can directly bind to NCOR2. (A) Effect of concentration of emodin and emodin-biotin (B-Emodin) on transcription of SerRS expressed as firefly/renilla ratio (n=3). (B) The protein level of SerRS with emodin (10 µM) and B-Emodin (10 µM). (C) SDS-PAGE of MDA-MB-231 cell lysates incubated with free biotin or B-Emodin following protein affinity pull-down and coomassie blue staining (n=3). Arrows indicate bands analyzed by mass spectrometry (MS). (D) Immunoblot with anti-NCOR2, anti-ACACA, anti-FASN, anti-CAD, and anti-α-Tubulin antibodies of protein precipitated by streptavidin beads from MDA-MB-231 cell lysates in the presence of B-Emodin or biotin (10 µM). (E) shRNA target human NCOR2 was transfected into MDA-MB-231 cells, NCOR2 expression in MDA-MB-231 cells was determined by qPCR after shRNA transfection. (F) mRNA levels of SerRS from MDA-MB-231-shLacZ and MDA-MB-231-shNCOR2 cells treated with emodin for 48 h (n=3). (G) Representations of the predicted binding modes of emodin with NCOR2, SANT2. (H) Immunoblot with anti-NCOR2 antibody of purified proteins (GST and GST-NCOR2 SANT2) incubated with B-Emodin (10 µM) or biotin and precipitated by streptavidin beads. Data are means ± SD., ns, no significant difference; **p<0.01; ***p<0.001; by one-way ANOVA for A; by Student's t-test for E, F.