XB-IMG-158343
Xenbase Image ID: 158343
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Figure S2, related to Figure 1: Purified Flag-M18BP1-2747-944 recapitulates cell
cycle regulated CENP-A nucleosome binding
A) M18BP1 binding of CENP-A chromatin is restricted to interphase. CENP-A chromatin
binding of full-length in vitro translated Flag-M18BP1-2 was assayed in either metaphasearrested
or interphase egg extracts depleted (Δ) or not depleted (+) of CENP-C. CENP-Cindependent
inhibition of M18BP1 binding was observed specifically in metaphase extract.
B) Coomassie stained SDS-PAGE gel of 500 ng of purified recombinant Flag-M18BP1-2747-944
wild-type or Y770A, W771A.
C) Flag-M18BP1-2747-944 wild-type or YW mutant binding to CENP-A or H3 chromatin. Wildtype
Flag-M18BP1-2747-944 binds specifically to CENP-A chromatin; mutation of the CENP-C
motif abolishes binding. Values are normalized to the wild-type protein binding to CENP-A
beads. Plot shows mean ± SEM from three experiments.
D) The CENP-A chromatin binding domain of M18BP1 recapitulates interphase-specific
chromatin binding. Representative images of purified Flag-M18BP1747-944 protein binding to
CENP-A chromatin in interphase or metaphase-arrested Xenopus egg extracts. Cell cycle state
indicated at left; immunolocalized protein indicated above. Scale bar, 5 μm.
E) Modification of CENP-A chromatin in extract does not regulate M18BP1 binding. CENP-A
chromatin was incubated in buffer, metaphase-arrested or interphase egg extract. After
incubation, CENP-A chromatin was re-isolated, and then incubated with Flag- M18BP1-2747-944
protein. Bar graph represents quantification of bound M18BP1 protein normalized to protein
bound to chromatin without extract incubation. 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 |