Fig. 1

Over-expression of 6 × His-tagged H1 Variants Does Not Alter Global Chromatin Structure. a U2OS Tet/On cells were transfected with plasmids encoding 6 × His-tagged human histone H1 variants. These cells were incubated 72 h with or without doxycycline before extracted. Whole cell extracts were resolved by SDS-PAGE gel, and immunoblotted with anti-His antibodies. b Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 and H1x) were incubated with doxycycline for 72 h. Whole cell, cytosolic and nuclear extracts of these cells were resolved by SDS-PAGE gels and visualized by western blotting with antibodies targeting proteins indicated on the right. c Total histones were extracted from untransfected (UN) U2OS Tet/On cells or Doxycycline-induced U2OS cells expressing 6 × His-tagged H1 variants (H1.1, H1.2 and H1x) using acid precipitation method. 30 μg of each sample was resolved by SDS-PAGE gel, and visualized by coomassie staining or immunoblotting with anti-His antibodies. d–f Nuclei from uninduced (UN) or Doxycycline induced (Dox) U2OS cells transfected with vectors expressing H1.1 (d) , H1.2 (e) or H1x (f) were digested with 0.2 U/ml (Sigma units) of MNase at 37 °C for various lengths of time, then quenched with EDTA. Digested DNA samples were purified using phenol extraction and ethanol precipitation, and resolved on 1 % agarose gel with EtBr staining