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

. 2000 Oct 16;151(2):289–296. doi: 10.1083/jcb.151.2.289

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

© 2000 The Rockefeller University Press

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 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

TRAPP subunits bind preferentially to the nucleotide-free form of Ypt1p. (A) A yeast lysate was incubated with agarose beads containing either GST (lane 1) or GST-Ypt1p (lanes 2–4) as described in the Materials and Methods. Before the incubation, Ypt1p was either stripped of nucleotide (nucleotide-free, lane 2) or loaded with GDP (lane 3) or GTPγS (lane 4). The beads were washed and the bound proteins were eluted by boiling in SDS-PAGE sample buffer. The eluate was then fractionated on a SDS–12.5% polyacrylamide gel, and Western blot analysis was performed by the enhanced chemiluminescence method using anti-Trs33p antibody at 1:2,500 dilution (top) or anti-Trs20p antibody at 1:1,000 dilution (bottom). (B) A yeast lysate was incubated with agarose beads containing either GST (lane 1), GST-Ypt1p (lanes 2 and 4), or GST-Ypt51p (lane 3). Before the incubation, Ypt1p and Ypt51p were stripped of nucleotide (nucleotide-free, lanes 2 and 3) or stripped of nucleotide and allowed to rebind GDP (lane 4). The beads were processed as above. The amount of Trs33p present in 0.1% of the lysate that was incubated with the beads is shown.

TRAPP subunits bind preferentially to the nucleotide-free form of Ypt1p. (A) A yeast lysate was incubated with agarose beads containing either GST (lane 1) or GST-Ypt1p (lanes 2–4) as described in the Materials and Methods. Before the incubation, Ypt1p was either stripped of nucleotide (nucleotide-free, lane 2) or loaded with GDP (lane 3) or GTPγS (lane 4). The beads were washed and the bound proteins were eluted by boiling in SDS-PAGE sample buffer. The eluate was then fractionated on a SDS–12.5% polyacrylamide gel, and Western blot analysis was performed by the enhanced chemiluminescence method using anti-Trs33p antibody at 1:2,500 dilution (top) or anti-Trs20p antibody at 1:1,000 dilution (bottom). (B) A yeast lysate was incubated with agarose beads containing either GST (lane 1), GST-Ypt1p (lanes 2 and 4), or GST-Ypt51p (lane 3). Before the incubation, Ypt1p and Ypt51p were stripped of nucleotide (nucleotide-free, lanes 2 and 3) or stripped of nucleotide and allowed to rebind GDP (lane 4). The beads were processed as above. The amount of Trs33p present in 0.1% of the lysate that was incubated with the beads is shown.