Association of SMAD2/3 with the megalin promoter.
(A) Schematic representation of the human megalin promoter (-1500 bp), highlighting the putative SBEs used to analyse megalin promoter activity. (B) HEK293 cells were seeded onto 10 cm culture dishes and transfected with the megalin promoter construct (-1500PromBasic) or the PAI-1 promoter construct (p800Luc) 48 h later. Co-transfections were performed in the presence or absence of exogenous FLAG-SMAD2 and FLAG-SMAD3 plasmids. After 24 h, the transfected cells were treated with 5 ng/ml TGF-ß1 for 1 hour and subjected to ChIP as described in the experimental section. The immunoprecipitated DNA was queried using negative control primers, a non-specific primer set and specific primers to the SBE-57 or SBE-605 sites of the megalin promoter and the SBE-280 or SBE-680 sites of the PAI-1 promoter (positive control). PCR was performed using DNA that was immunoprecipitated using an HA antibody as a negative control (HA lane). The PCR from the input DNA served as another positive control (input lane). The products were analysed by agarose gel electrophoresis. (C) The qPCR signals derived from the ChIP samples were divided by the qPCR signals derived from the input sample collected earlier during the ChIP procedure. The relative fold change was calculated from the mean ± SD 2(^)ΔΔCT values (n = 2), as described in the experimental section, and normalized with the input.