Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2017 Mar 6;216(3):607–621. doi: 10.1083/jcb.201608083

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2017 Nechemia-Arbely et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 4. — Affinity purified CENP-A^LAP expressed from an endogenous CENP-A locus is assembled into chromatin as a homotypic octamer and does not contain H3. (A) Schematic for construction and use of a HeLa cell line in which CENP-A was tagged at the endogenous allele with a LAP-tag consisting of Hisx6, a Precission protease cleavage site and EYFP (Mata et al., 2012). The resulting CENP-A^+/LAP cell line was adapted to growth in suspension. (B) Localization of endogenously tagged CENP-A^LAP determined with indirect immunofluorescence using anti-GFP antibody. Bars: 5 µm; (inset) 1 µm. (C) Experimental design for affinity purification of endogenously tagged CENP-A^LAP monochromatin particles. (D) SDS-PAGE of serial 1:2 dilutions of input, immunoprecipitation, and unbound fractions of CENP-A^LAP affinity purification from cells synchronized in G2. (E, left) Quantification of CENP-A^LAP and nontagged CENP-A levels in the blot shown in D before correcting for the fraction of CENP-A that passes through the membrane. (right) Quantification of CENP-A^LAP and nontagged CENP-A levels in the input and immunoprecipitation (IP) in the blot shown in D after correcting for the underscoring of CENP-A as shown in Fig. S3 (A and B). For each panel, n = 3 from three independent loadings in the gel shown in D. Error bars represent SEM. (F) CENP-A^LAP immunoprecipitation efficiency (left) and levels of unbound nontagged endogenous CENP-A (right) were quantified in the blot shown in D. For each panel, n = 3 from three independent loadings in the gel shown in D. Error bars represent SEM.