The H3-MTA interaction independent mechanism exists for NURD chromatin association. A. Pulldown assay using GFP-tagged MTA1 proteins confirmed the MTA1 C-terminal H3 binding activity. Note that a very weak H3 binding activity was detected for N-terminal MTA1 with intensive exposure. B. Both the N-terminal and C-terminal GFP-MTA1 were chromatin associated in 293T cells. The 293T cells were transfected with GFP-MTA1, GFP-MTA1(1–460) and GFP-MTA1(454–715) and the resulting cells were subjected to cellular fractionation of soluble cellular fraction and insoluble chromatin fraction as described in Materials and Methods. The fractions were further extracted with buffer containing different concentration of NaCl as indicated to generate different fractions. Note that while DNMT1 was gradually stripped from chromatin by an increasing salt concentration, all three MTA1 proteins essentially remained to be associated with chromatin. C. Fractionation of soluble chromatin fragments derived from MNase digestion by a 5–30% sucrose gradient centrifugation. The soluble chromatin was prepared from the 293T cells expressing both GFP-MTA1(1–460) and GFP-MTA1(454–715). After sucrose gradient centrifugation, consecutive fractions (300 μl each) were collected from top. Half of the samples were used for preparation of DNA and analyzed by agarose gel electrophoresis. D. The remaining half of the sucrose gradient fractionation samples was subjected to protein precipitation by TCA and resolved by SDS-PAGE. After transfer to nitrocellulose membrane, the proteins were revealed by Ponceau S staining (top panel) and then analyzed by western blot. Note that the majority of GFP-MTA1(1–460) and GFP-MTA1(454–715) were present in the chromatin fractions.