FIG. 4.

Expression of an enzymatically inactive HDAC1 mutant induces a subset of HDAC1 target genes. (A) Left panel, Western blot analysis of whole-cell extracts prepared from HDAC1-H140/141A mutant-expressing HDAC1 KO cells (KO-mut) and respective wild-type (WT-vec) and KO (KO-vec) controls. The blot was incubated sequentially with antibodies specific for HDAC1, HDAC2, and β-actin, respectively. Right panel, equal amounts of the extracts described for the left panel were analyzed for total deacetylase activity. Data presented for relative HDAC activities are mean values for three independent experiments. (B) Left panel, Western blot analysis of HDAC2 immunoprecipitates prepared from the protein extracts described for panel A with antibodies specific for HDAC1 and HDAC2. Right panel, comparison of HDAC2-associated deacetylase activities after immunoprecipitation with an HDAC2-specific antibody from the whole-cell extracts described for panel A. Data presented are representative of three independent experiments. (C) SYBR green real-time RT-PCR analysis of target genes. Total RNA isolated from KO-mut cells and vector-transfected KO cells was analyzed by quantitative RT-PCR for the expression of the indicated genes. Expression levels (n-fold) were normalized to expression levels in KO-vec cells and presented as mean values for three experiments.