Xenbase Image ID: 158338

Figure 1 Xenopus M18BP1 Directly Binds CENP-A Chromatin (A) Schematic of in vitro chromatin binding assay. Reconstituted CENP-A or H3 chromatin, immobilized on streptavidin-coated beads, was incubated in rabbit reticulocyte lysate containing M18BP1. Following chromatin isolation, M18BP1 binding was assayed by immunofluorescence. (B) Both isoforms of Xenopus M18BP1 bind selectively to CENP-A chromatin. Representative immunofluorescence images of M18BP1 binding to CENP-A chromatin (top) and H3 chromatin (bottom), both labeled with Myc-tagged histone H4. The M18BP1 isoform is indicated to the left; immunolocalized protein is indicated above. Scale bar, 5 μm. (C) (Left) Quantification of (B). Values are normalized to M18BP1-2 signal on CENP-A beads. (Right) Representative western blot of samples from (B). Top portion (anti-FLAG) shows similar translation of M18BP1 isoforms; bottom portion (Ponceau stained) is a loading control. Asterisk (∗) indicates an M18BP1 break-down product. (D) Schematic representation of truncations of M18BP1-2 used to define the CENP-A chromatin binding domain (magenta) in (E) and (F). (E and F) M18BP1-2 binds CENP-A nucleosomes via amino acids 747–944. M18BP1-2 truncations spanning the indicated amino acids were assayed for CENP-A chromatin binding by the method described in (A), Values are normalized to full-length M18BP1-2 signal. All graphs show the mean ± SEM of at least three experiments. See also Figures S1–S3. Image published in: French BT et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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