Figure 5. Prohibitin is required to recruit PIP2 and HDAC1 to the promoters of WT1 target genes.

(A) U2OS cells were transfected with control siRNA, BASP1 siRNA or prohibitin siRNA. 48hr later the cells were subjected to ChIP assay with control IgG antibodies, PIP2 antibodies and HDAC1 antibodies. The results are presented as fold enrichment at AREG and cmyc promoter relative to a control genome region. Error bars are presented as the SDM of three independent experiments and *p<0.05 by Student’s t test. (B) Control V-K562 cells, wild type BASP1-K562 cells and the mutant derivate G2A BASP1-K562 cells were subjected to ChIP with control IgG antibodies, BASP1 antibodies and prohibitin antibodies at AREG promoter relative to a control genome region. Error bars are the SDM of three independent experiments and *p<0.05 by Student’s t test. Lower panel; Nuclear extracts from BASP1-K562 cells and BASP1 G2A-K562 cells were used in immunoprecipitation with anti-prohibitin antibodies. The immunoprecipitates were immunoblotted with anti-BASP1 antibodies. (C) U2OS cells were transfected with BASP1 siRNA, prohibitin siRNA or a control siRNA and 48 hr later the cells were subjected to ChIP with control IgG antibodies and anti-CBP antibodies. The results are presents as fold enrichment at the AREG and c-myc promoter regions compared to a control genome region. Error bars are SDM of three independent experiments (*p<0.05 by Student’s t test). In parallel, whole-cell extracts were immunoblotted with anti-BASP1, anti-prohibitin or anti-tubulin antibodies to confirm knockdown of BASP1 or prohibitin.