Figure 4. Human HJURP promotes loading... | Rockefeller University Press

Figure 4.

$Figure 4. Human HJURP promotes loading of Xenopus CENP-A. (A) Outline of the chromatin assembly experiment. Mitotic chromosomes (M) and interphase nuclei (I) were assembled in mock- and CENP-A–depleted extracts containing in vitro–translated (IVT) myc-CENPA. (B) Nuclei and chromosomes were stained with antibodies against myc (top) and CENP-A (second from top), both color coded with a gradient as in Fig. 1 B. Insets 1–4 show that myc signals (red) overlap with CENP-A signals (green) in interphase nuclei of mock-depleted extracts (inset 2 and 6), whereas weak myc signals in CENP-A–depleted interphase nuclei (inset 4) are mostly background. DAPI staining (red) and biotin-dUTP (cyan) are shown in the bottom row. Bar, 10 µm. (C) IVT myc-CENP-A was added to a CENP-A–depleted extract along with sperm DNA and samples of the reaction were taken at the indicated times and analyzed by immunoblot with anti-myc and anti-RbAp48 as loading control. The time of calcium addition is considered t = 0. Cell cycle progression was monitored by chromosome morphology after DAPI staining (not depicted). (D) Immunoblot analysis of the extracts used for chromatin assembly in panel E. Antibodies against human and Xenopus HJURP were used to detect GST-HJURP (top) and endogenous xHJURP (bottom), respectively, whereas anti-CENPA was used to simultaneously detect myc-CENPA and endogenous CENP-A (middle). (E) Representative images of interphase nuclei from the indicated extracts stained with anti–CENP-A (green) and DAPI (red). Insets in the images of the first row show biotin-dUTP incorporation (cyan). In the images from the middle and bottom rows, CENP-A labeling has been color coded as in Fig. 1 B. In the bottom row, all centromeres from the nuclei presented above are shown at higher magnification. Bar, 10 µm. (F) Graph showing the loading efficiency of CENP-A in the indicated extracts compared with a mock-depleted extract. Bars represent mean ± SE from two independent experiments. (G) GST-HJURP interacts with myc-CENPA and endogenous CENP-A. Analysis of an immunoprecipitation reaction with anti-GST from an egg extract containing myc-CENPA and either GST alone or GST-HJURP. Aliquots (1.5%) from input and flow-through (FT) fractions were also analyzed.$

Human HJURP promotes loading of Xenopus CENP-A. (A) Outline of the chromatin assembly experiment. Mitotic chromosomes (M) and interphase nuclei (I) were assembled in mock- and CENP-A–depleted extracts containing in vitro–translated (IVT) myc-CENPA. (B) Nuclei and chromosomes were stained with antibodies against myc (top) and CENP-A (second from top), both color coded with a gradient as in Fig. 1 B. Insets 1–4 show that myc signals (red) overlap with CENP-A signals (green) in interphase nuclei of mock-depleted extracts (inset 2 and 6), whereas weak myc signals in CENP-A–depleted interphase nuclei (inset 4) are mostly background. DAPI staining (red) and biotin-dUTP (cyan) are shown in the bottom row. Bar, 10 µm. (C) IVT myc-CENP-A was added to a CENP-A–depleted extract along with sperm DNA and samples of the reaction were taken at the indicated times and analyzed by immunoblot with anti-myc and anti-RbAp48 as loading control. The time of calcium addition is considered t = 0. Cell cycle progression was monitored by chromosome morphology after DAPI staining (not depicted). (D) Immunoblot analysis of the extracts used for chromatin assembly in panel E. Antibodies against human and Xenopus HJURP were used to detect GST-HJURP (top) and endogenous xHJURP (bottom), respectively, whereas anti-CENPA was used to simultaneously detect myc-CENPA and endogenous CENP-A (middle). (E) Representative images of interphase nuclei from the indicated extracts stained with anti–CENP-A (green) and DAPI (red). Insets in the images of the first row show biotin-dUTP incorporation (cyan). In the images from the middle and bottom rows, CENP-A labeling has been color coded as in Fig. 1 B. In the bottom row, all centromeres from the nuclei presented above are shown at higher magnification. Bar, 10 µm. (F) Graph showing the loading efficiency of CENP-A in the indicated extracts compared with a mock-depleted extract. Bars represent mean ± SE from two independent experiments. (G) GST-HJURP interacts with myc-CENPA and endogenous CENP-A. Analysis of an immunoprecipitation reaction with anti-GST from an egg extract containing myc-CENPA and either GST alone or GST-HJURP. Aliquots (1.5%) from input and flow-through (FT) fractions were also analyzed.

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