Anti ar n 20 sc 816

HepG2 cells were serum-starved for 48 h, as above, and then treated for 1, 3, or 6 h with DMSO (vehicle control), 10 nM E₂, 10 nM DHEA, or 10 nM DHT before crosslinking with 1% formaldehyde for 5 min. ChIP was performed using MAGnify ChIP (Invitrogen). Lysates were incubated with anti-AR (N-20, sc-816, Santa Cruz), anti-ERα (HC-20, sc-543, Santa Cruz), anti-ERβ (HC-150, sc-8974, Santa Cruz), or mouse IgG (Invitrogen). Immunoprecipitated DNA was amplified by quantitative PCR using the following primers for the miR-21 promoter : ERE1-F: 5’-CCAGAAGTTAGGGATATGTTAGCA-3’; ERE1-R: 5’-TACCTCCAGGGTTCAAGTGATTCT-3’; ChIP negative controls (12,038 at 3’ end of VMP1/TMEM49): Neg-F: 5’-ATTGGCTATCTTTGTGTGCCTTG-3’; Neg-R: 5’-TGCTCAATAAAACACATTGTTCTTCAT-3’; ARE-1F: 5’-TCCCAATCATCTCAGAACAAGCT-3; ARE-1R: 5’-TGCACAGAAACTCCAGTACATTAGTAAC-3’; ARE-2F 5’-GGATGACGCACAGATTGTCCTA-3’; ARE-2R: 5’-AAAGAAACTGCCCGCCCTCT-3’. Each ChIP was performed in triplicate, and each PCR was amplified in triplicate. Quantitation was performed as described in (Mattingly et al., 2008 (link)). For IP of ERα, ERβ, and AR with IgG, CT was “undetermined”; for ERβ with ERE1 in DHEA- and E₂- treated cells (3 h), CT values were 32.1 ± 0.51 and 34.2 ± 0.51, respectively.