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

. 1997 Apr 21;137(2):305–317. doi: 10.1083/jcb.137.2.305

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

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

G_α and G_β are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100 μCi of [³⁵S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from the supernatant were done either in the presence (+) or absence (−) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover α subunits and β was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A–C). Arrows indicate the positions of β and α subunits. (B) IMR-32 cells were labeled for 2 h with 250 μCi [³⁵S]methionine followed by solubilization in NP-40/ Lubrol lysis buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of G_β subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 μCi [³⁵S]methionine and chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 μCi [³⁵S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114–containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (−) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins. Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows on the right indicate the appearance of some of the proteins specifically released by PI-PLC.

G_α and G_β are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100 μCi of [³⁵S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from the supernatant were done either in the presence (+) or absence (−) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover α subunits and β was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A–C). Arrows indicate the positions of β and α subunits. (B) IMR-32 cells were labeled for 2 h with 250 μCi [³⁵S]methionine followed by solubilization in NP-40/ Lubrol lysis buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of G_β subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 μCi [³⁵S]methionine and chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 μCi [³⁵S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114–containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (−) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins. Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows on the right indicate the appearance of some of the proteins specifically released by PI-PLC.