Figure 3.
ELYS Chromatin Association Directs Nuclear Pore Complex Assembly
(A) Sperm chromatin was incubated in complete or membrane-free egg extract for 60 min. Chromatin was isolated and immunoblotted for ELYS, Nups133, 358, 214, 153, 62, and Mcm2. The lower portion of the gel was stained with Coomassie blue so that the level of histones could be determined as a control for chromatin recovery.
(B) rATH was incubated in the presence or absence of sperm chromatin for 30 min. Chromatin was isolated and immunoblotted for rATH. The lower portion of the gel was stained with Coomassie blue so that the level of histones could be determined as a control for chromatin recovery. Twenty-five percent of input rATH is shown as control for protein recovery.
(C) Sperm chromatin was incubated for 90 min in egg extract treated with either buffer alone or rATH at the indicated concentrations. Chromatin was isolated and immunoblotted for rATH, ELYS, Nups133, 358, 214, 153, 62, and Mcm2. The lower portion of the gel was stained with Coomassie blue so that the level of histones could be determined as a control for chromatin recovery.
(D) DNA synthesis was assayed in rATH-treated extracts.
(E) Sperm chromatin was incubated in extract treated with 10 μg/ml DilC18 (red) and either buffer or 2 μM rATH. Nuclei were fixed and spun onto coverslips and counterstained with DAPI (blue). The scale bar represents 10 μm.
(F) Nuclei assembled in egg extract treated with buffer alone or 10μM rATH were fixed, spun onto coverslips and stained with mAb414 (green) and DAPI (blue).