XB-ART-53863Dev Cell January 1, 2017; 42 (2): 190-199.e10.
Xenopus laevis M18BP1 Directly Binds Existing CENP-A Nucleosomes to Promote Centromeric Chromatin Assembly.
PubMed ID: 28743005
PMC ID: PMC5544353
Article link: Dev Cell
Grant support: R01 GM074728 NIGMS NIH HHS , T32 GM007276 NIGMS NIH HHS
Genes referenced: cenpa cenpc cenpn mis18bp1 myc
Antibodies referenced: Mis18bp1 Ab1
Article Images: [+] show captions
|Figure S3, related to Figure 1 and S2: Mass spectrometry identification of putative regulatory residues in M18BP1 CENP-A nucleosome binding domain A) Flag-M18BP1-2747-944 or MBP-Flag-M18BP1-2747-944 binding to CENP-A chromatin was assayed in metaphase-arrested and interphase Xenopus egg extract. The MBP tag did not affect metaphase inhibition of CENP-A nucleosome binding, although it did prevent some CENP-A nucleosome binding in interphase. Values are normalized to Flag-M18BP1-2747-944 binding in interphase extract. B) Coomassie gel of MBP-M18BP1-2747-944 immunoprecipitated from metaphase-arrested Xenopus egg extract with anti-MBP. The boxed band was submitted for mass spectrometry analysis for post-translational modifications. C) Table displaying all phosphorylation events found on M18BP1-2747-944 by mass spectrometry after incubation of MBP-M18BP1-2747-944 in metaphase egg extracts. Green highlighting indicates the phosphorylated residue forms a minimal Cdk consensus motif (S/T-P). Estimated abundance of each modification is indicated. D) Translated full-length M18BP1-2 containing mutations in modification site residues were assayed for in vitro binding to CENP-A chromatin by immunofluorescence. Values are normalized to wild-type M18BP1-2 signal on CENP-A beads. Plot shows the mean ± SEM of at least three experiments. E) Representative anti-Flag Western blots of reactions from D showing equivalent translation of all Flag-M18BP1-2 mutants.|