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

. 2003 Feb 17;160(4):529–539. doi: 10.1083/jcb.200210095

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

Copyright © 2003, 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

Figure 2. — Purification of TrAF and identification as the TRAP complex. (A) Shown is a Coomassie blue–stained gel of fractions resulting from the separation of membrane proteins by ion exchange and ConA chromatography. The positions of CNX and the α through δ subunits of the TRAP complex are indicated. (B) Immunoblots against various proteins in proteoliposomes prepared using the fractions in panel A. Lanes 1 and 2 contain proteoliposomes reconstituted from the total and Q-depleted detergent extracts, respectively. Lanes 3–12 contain proteoliposomes coreconstituted with the Q-depleted detergent extract plus the respective individual fractions in lanes 3–12 of A. (C) Translocation assays of PrP and Prl using proteoliposomes from panel B. Control reactions lacking membranes or containing RMs are also shown for comparison. Only the translocated material, remaining after digestion of the translation reactions with PK, is shown. The efficiencies of ^secPrP and Prl translocation, relative to the unfractionated proteoliposomes in lane 1, are shown below the autoradiograph. (D) Purification of the TRAP complex from the RAMP fraction. Shown is the Coomassie blue–stained gel containing the final fractions of the purification. (E) A Q-depleted detergent extract was replenished with varying concentrations of RAMP-purified TRAP or total Q eluate and reconstituted into proteoliposomes. As a control, an unfractionated detergent extract was also reconstituted in parallel. Shown in the top panel is an immunoblot against TRAPα of the different proteoliposomes. The amount of TRAP, as a percent of that found in the unfractionated proteoliposomes, is indicated above the blot. The bottom panel shows the assay for PrP translocation into these proteoliposomes. Only the translocated products remaining after protease digestion are shown. (F) The extent of ^secPrP translocation in the assay from panel E was quantitated and plotted as a bar graph. The amount of translocation in the Q-depleted proteoliposomes replenished with the total Q-eluate fraction was defined as 100%.