Figure 3

The lethal phenotype-defective IN mutants lack chromatin binding ability. A. Chromatin binding ability of IN in yeast cells. After growing in IN-inducible media for 3h, different IN expressor-transformed yeast cells were lysed. Whole cell extracts were incubated with 50 or 200mM NaCl for 20 min before the separation of chromatin-bound and non-chromatin-bound fractions. Both fractions were subjected to WB using anti-IN antibody (left panel). Right panel: The INwt-expressing yeast cells were fractionated into chromatin- and non-chromatin-bound fractions and the presence of the Lys20/21 protein was detected by WB using anti-Lys20.21 antibody. B. 293T cells transfected with different HA-tagged-IN expressors were lysed in cold CSK I buffer (0.5% Triton X-100), fractionated and the presence of HA-IN in the chromatin-bound and non-chromatin-bound fractions was analyzed by IP and WB with anti-HA antibodies (left panel). In parallel, the presence of the nuclear pore complex-associated protein Nup62 or YFP in different fractions of 293T cells or cells transfected with a SVCMV-YFP expressor was also analyzed by using anti-Nup62 and anti-GFP antibodies (right panel). S1: Supernatant (non-chromatin-bound fraction); P1: Pellet (chromatin-bound fraction); S2: DNase-released chromatin-associated proteins in P1; P2: insoluble, cytoskeletal, and nuclear matrix proteins in P1.