xHJURP is required for CENP-A deposition. (A) Left, immunoblot analysis of increasing amounts of a mock-depleted extract (5–100% of a 1.5-µl aliquot, lanes 1–5) and a 1.5-µl aliquot of an xHJURP-depleted extract (lane 6). Right, graph showing the loading efficiency of CENP-A in an extract depleted of xHJURP. Data come from two different experiments. Bars represent mean ± SE. (B) Immunoblot analysis of mock-depleted and xHJURP-depleted extracts replenished or not with myc-CENP-A and GST-HJURP at the time of sperm addition (t = 0, lanes 1–6) and at the end of the experiment (t = 3 h, lanes 7–12). (C) Loading efficiency of CENP-A in the indicated extracts. Data come from two independent experiments. Bars represent mean ± SE. (D) Outline of the chromatin assembly reaction used to assess the localization of xHJURP throughout the cell cycle reproduced in the extract by successive additions of calcium and CSF extract. Sperm chromatin (800/µl) was added at time 0. The times at which aliquots were taken for analysis are indicated. (E) Immunofluorescent staining with anti-xHJURP (green), CENP-A (red), and DAPI (blue). White arrowheads point to HJURP signals that colocalize with CENP-A signals on CSF assembled chromosomes. Bar, 10 µm.