MacroH2A1 knockdown cell lines by siRNA. (A) MacroH2A1 protein levels in knockdown cell lines. Histone extracts were prepared from six different types of Neuro2A cells: those were not transfected (NT), transfected with the empty pSicoR vector (pSicoR), or transfected with macroH2A1 siRNA constructs (#2, #4, #10 and #11). These histone extracts (5 μg per each well) were separated on 14% SDS-PAGE and analyzed with western blotting (Top panel). Even loading and transfer of the histone extracts was monitored by staining with Coomassie Blue (Bottom panel). (B) Chromatin immunoprecipitation analysis of macroH2A1 in the differentially methylated regions (DMRs) and promoters of several imprinted genes. IG-DMR (Intergenic DMR) is an ICR located in the Dlk1/Gtl2 imprinted domain whereas H19-ICR is an ICR located 2-kb upstream of H19. Individual ChIP analyses were performed using the three types of cells: without transfection (NT) and the two stable transfectants (#2 and #11). The amplified PCR products derived from immunoprecipitated DNAs were compared with those amplified from the chromatin DNA before immunoprecipitation (Input, 10%). Since three individual ChIP experiments derived similar amounts of Input DNAs, a representative Input from an NT sample is shown.