Mapping of the interaction domains between PLZF and N-CoR. (A) Coimmunoprecipitation of N-CoR and PLZF. PML and PLZF were radiolabeled and incubated with in vitro translated N-CoR. After immunoprecipitation with anti–N-CoR antibody (αN-CoR), proteins were analyzed by SDS-PAGE and autoradiography. Twenty-five percent of input is shown (input). (B) N-CoR mutants (as indicated by amino acids numbers) were labeled in vitro with ³⁵S-methionine, incubated with GST-PLZF affinity matrix, and analyzed in pulldown assays. (C) Partial N-CoR proteins (delineated by amino acid numbers) were expressed in bacteria as GST fusions and used in pulldown assays for in vitro interaction with radiolabeled PLZF. Ten percent of input is shown (input). (D) Various PLZF mutants, as indicated, were radiolabeled and incubated with GST-N-CoR affinity matrix (amino acids 1829-1940, containing the C-terminal Sin3 interaction domain). PLZF▵POZ and 5′PLZF correspond to PLZF without the BTB/POZ domain and the region of PLZF contained in the PLZF-RARα chimeric protein, respectively. Ten percent input is shown (input). (E) Radiolabeled Sin3A and B proteins were incubated with GST-N-CoR (amino acids 1679-2453), GST-PLZF, or GST affinity matrix and analyzed in pulldown assays. Twenty-five percent input is shown (input). (F) HDAC1 was labeled in vitro with ³⁵S-methionine and subjected to either the pulldown assay with GST-PLZF or coimmunoprecipitation with in vitro translated Sin3A protein and anti-Sin3A antibody (αSin3A). Twenty-five percent input is shown (input).