FIG. 5.

SRY binding to Ntf3 promoter. A) In vitro transcribed and translated SRY protein with HA tag was subjected to binding with a 300-bp Ntf3 promoter fragment containing either intact or mutated SRY/SOX binding sites. An anti-HA antibody immunoprecipitated the Ntf3 promoter fragment containing the SRY binding site, but not the mutated one. Nonimmune IgG was used as a negative control. Bands indicate PCR products amplified by rat Ntf3 promoter-specific primer flanking SRY binding site. Lanes are: Water (H₂O); non-immune IgG (negative control; IgG); Ntf3 promoter with intact SRY binding site (SRY (Intact)); Ntf3 promoter with mutated SRY binding site (SRY (Mutated)); positive control for PCR (genomic DNA). Representative of a minimum of three experiments. B and C) In vivo SRY binding used a cChIP assay. Chromatin from 20 pairs of testes were used for immunoprecipitation with anti-SRY antibodies, Koopman antibody (B) and commercial Santa Cruz antibody (C), followed by centrifugation. Immunoprecipitated DNA was purified with a phenol chloroform method and used for PCR. Two rounds of PCR reaction were used to get the expected band, which was later sequence verified. Nonimmune IgG was used as a negative control. Bands indicate PCR products amplified by rat Ntf3 promoter-specific primer flanking SRY binding site for water control (H₂O); nonimmune IgG (IgG); input chromatin DNA (Input); anti-SRY antibody ChIP (αSRY). Representative of a minimum of three different experiments.