Figure 1

An assay to measure CENP-A incorporation in Xenopus egg extracts. Step 1: Extract preparation and depletion. Extracts are prepared from laid eggs arrested in metaphase II (mitotic extracts) and subjected to immunodepletion with specific antibodies against factor X. Step 2: Nuclei assembly. Sperm chromatin is added to mitotic extracts in two different tubes. In one tube (top), incubation proceeds for 80-120 min to get mitotic chromosomes. In another tube (bottom), calcium is added to drive entry into interphase 40 min after sperm addition. Incubation proceeds for 80 min to get interphase nuclei that have undergone replication. Step 3: Nuclei isolation. Equal volumes of the reaction mixtures in both tubes are combined, fixed and centrifuged over a coverslip placed at the bottom of a glycerol cushion. Step 4: Immunofluorescence. Coverslips are processed for immunofluorescence with an antibody against CENP-A and DNA is stained with DAPI. Step 5: Image acquisition and analysis. Images of a mass of mitotic chromosomes next to an interphase nucleus are acquired and CENP-A signals are quantitated using ImageJ software (http://rsb.info.nih.gov/ij/). The average Integrated Density (ID = average pixel intensity × area) is first calculated from the IDs of individual centromeres within each interphase nucleus (ID_I) and neighboring mass of mitotic chromosomes (ID_M) and then a ratio between the IDs of each imaged pair is obtained (IDr). Finally, the average ID ratio (IDr) of at least 15 pairs is calculated. The relative CENP-A loading efficiency of a depleted extract with respect to a mock depleted extract (considered 100%) is calculated and represented. If depletion of a factor X prevents loading of CENP-A and the defect is rescued by adding back the factor to the depleted extract, we conclude that factor X is involved in CENP-A incorporation.