FIG. 8.

PEX1 is a transcriptional regulator of the ANF promoter. (A) EMSA was performed with nuclear extracts prepared from cardiomyocytes on PERE and SP1 probes. Binding disruption with antibodies to PEX1 (αPEX1), SP1 (αSP1), or immunoglobulin G were used to confirm the identity of the PERE (closed arrowheads) and SP1 (open arrowhead) DNA-binding complex in cardiomyocytes. (B and C) Binding of bacterially produced PEX1 on the PERE probe. In panel B, GST-PEX1 was used; competition with cold PERE (self) was done at a 100- to 500-fold excess, and antibody blocking was performed with increasing amount of the anti-PEX1 antibody (αPEX1). In panel C, increasing amounts of MBP-PEX1 or MBP-LacZ were used with the PERE probe; the close arrowheads indicate the position of the DNA-binding complexes specifically obtained with the recombinant PEX1 protein. (D) Cardiomyocytes were cotransfected with wild-type and mutant ANF luciferase reporters and increasing amounts of PEX1 expression vector. The data shown represent the mean ± the SEM of at least six independent determinations.