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

. 2011 Jan 1;22(1):91–104. doi: 10.1091/mbc.E10-06-0543

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

© 2011 Ruepp et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

PMC Copyright notice

FIGURE 10: — Interaction of CstF-64 and CstF-64Tau with CstF-77. (A) In vitro pull-down. A recombinant C-terminal Flag-tagged fragment of CstF-77 was incubated with in vitro–translated HA-CstF-64 or HA-CstF-64Tau and with α-Flag-agarose followed by subsequent washes and separation by SDS–PAGE. Agarose beads with BSA served as negative control. Coprecipitated proteins were detected by Western blot with α-HA-HRP. (B) In vitro binding competition. Procedures were as described for (A), except that mixtures of in vitro–translated HA-CstF-64 and HA-CstF-64Tau were used as binding substrates. The ratios indicated in brackets refer to the amount of individual template used in the coupled transcription/translation reaction. (C) Inverse in vitro binding competition. In vitro translation products from reactions A (Flag-CstF-64Tau, HA-CstF-77 [full-length], and HA-β-globin) or B (Flag-CstF-64Tau, HA-CstF-77 [full-length], and HA-CstF-64) were incubated with α-Flag-Agarose to precipitate Flag-CstF-64Tau, followed by washes and separation by SDS–PAGE. In the control reactions, the template for Flag-CstF-64Tau was omitted. The presence of Flag-CstF-64Tau and HA-CstF-64 in the reaction was confirmed by Western blot with 10% of the input samples by using an antibody detecting both proteins simultaneously (C-20 antibody, Santa Cruz). The presence of Flag-CstF-64Tau in the precipitate was confirmed by Western blot with 10% of the precipitate by using α-Flag-HRP. Coprecipitated HA-CstF-77 was detected with α-HA-HRP.