Figure 7.
Chromatin-bound ELYS/Nup107-160 complex recruit POM121- and NDC1-containing membrane vesicles. (A) Reconstituted nuclei were assembled in the presence of 15 μM GST, GST-AT-hook+, or GST-AT-hook-2R→A. The presence of POM121 in the nuclear membranes was probed for with directly labeled anti-POM121-AF488 (green). Membranes were stained with R18 membrane dye (red). DNA was stained with Hoechst (blue). Nuclei were fixed before mounting onto slides. Scale bar, 10 μm. (B) Reconstituted nuclei were assembled in the presence of buffer or 10 μm Distamycin A and processed as in A. Scale bar, 10 μm. (C) Anchored nuclei were assembled by incubating chromatin-coated coverslips with membranes and cytosol for 1 h, in the presence of either 15 μM GST, GST-AT-hook+, or GST-ΔAT-hook. A coverslip not treated with chromatin, but incubated with membranes, M, and cytosol, cyt, was used as a control for nonspecific binding (lane 4). The recruitment of integral membrane pore proteins NDC1 and gp210, in the presence (GST and GST-ΔAT-hook, lane 1 and 3) or absence (GST-AT-hook+, lane 2) of chromatin-bound ELYS/Nup107-160 complex, was determined by immunoblot analysis.