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. 1999 Aug 17;96(17):9873–9878. doi: 10.1073/pnas.96.17.9873

Figure 4.

Figure 4

CoREST exhibits repressor activity when fused to a Gal4- DBD and mediates repressor activity of REST. (a) Gal4-DBD (Gal4; amino acids 1–147) or Gal4CoREST expression vectors were transfected into HEK293 and PC12 cells, with a UAS type II CAT reporter gene (molar ratio of 1:1). (b Upper) Gal4REST lacking the N-terminal repressor domain (Gal4RESTΔN) was transfected into HEK293 cells along with a UAS type II CAT reporter gene. The role of CoREST was tested by addition of a cDNA coding for a REST competitor peptide containing the CoREST-binding site (Gal4RESTΔN + C3). Specificity of derepression was tested by over-expressing CoREST (Gal4RESTΔN + C3 + CoREST). (Lower) REST lacking the N-terminal repressor domain (RESTΔN) was transfected into PC12 cells along with an RE1 type II reporter gene. The role of CoREST was tested by addition of a cDNA coding for a REST competitor peptide containing the CoREST-binding site (RESTΔN + C3). Specificity of derepression was tested by overexpressing CoREST (RESTΔN + C3 + CoREST). CAT activity was monitored by TLC and autoradiography. Statistics were performed by using the nonparametric Mann–Whitney U test. ∗, P < 0.056 for RESTΔN + C3 +CoREST compared with RESTΔN + C3 and Gal4RESTΔN + C3 + CoREST compared with Gal4RESTΔN + C3; ∗∗, P < 0.0028 for RESTΔN + C3 compared with RESTΔN; ∗∗∗, P < 0.0008 for Gal4RESTΔN + C3 compared with Gal4RESTΔN. (c) Western blot of nuclear extracts of HEK293 cells transfected as in b. The probe was a monoclonal αGal4 antibody. (d) Western blot from nuclear (N) and cytoplasmic (C) extracts of HEK293 cells transfected with an HA-tagged C3. The probe was a monoclonal αHA antibody.