ChIP assay of the <i>CATSPER1</i> promoter at the SRY overlapping binding sites.

Immunoprecipitation of chromatin fragments was carried out with 1 μg of RNA polymerase II (RNAPol II), and SRY (α-SRY) and α-dystroglycan (α-mouse) antibodies in untransfected and pIRES-SRY transfected HEK-293 cells as well as in spermatogenic cells. Chromatin without antibody, the IgG mouse and the α-dystroglycan antibody were used as negative controls; RNAPol II and 1/10 of Input were used as positive controls. After reverse cross-linking, RNAse and proteinase K treatments were performed and the purified DNA samples were analyzed by PCR with oligonucleotides for each SRY site. As a negative control for PCR, water was added instead of the immunoprecipitated chromatin fragments (NC). A fragment from the BORIS promoter (GC-rich) was amplified as a negative control (unspecific SRY binding). Representative PCR amplifications for the different SRY sites are shown (lower panel; n = 3).