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Analysis of Plasma Membrane, Endosomal and Cytoplasmic fractions of mouse embryonic fibroblast

ABSTRACT: This project aimed to identify novel endosomal proteins and therefore cellular fractionation experiments were performed. Adherent mouse embryonic fibroblast were scraped on ice into hypotonic lysis buffer (10 mM HEPES-KOH pH 7.2, 0.25 M sucrose, 1 mM EDTA, 1 mM MgOAc and protease & phosphatase inhibitors (PhosSTOP and Complete mini tablets from Roche). Cell membranes were fragmented with French Press and nuclei removed with 10 min 1000 x g centrifugation. Plasma membrane fraction was collected with 10 min 10 000 x g centrifugation and endosomal/cytoplasmic fractions with 1h 100,000 x g centrifugation. Membrane fractions were washed at least once with lysis buffer and cytoplasmic fractions were centrifuged twice. All fractionation steps were performed at +4°C or on ice. All fractions were dissolved in sample buffer for immunoblotting

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryo, Fibroblast

SUBMITTER: Guillaume Jacquemet

LAB HEAD: Johanna Ivaska

PROVIDER: PXD001870 | Pride | 2015-10-14

REPOSITORIES: Pride

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Integrin endosomal signalling suppresses anoikis.

Alanko Jonna J Mai Anja A Jacquemet Guillaume G Schauer Kristine K Kaukonen Riina R Saari Markku M Goud Bruno B Ivaska Johanna J

Nature cell biology 20151005 11

Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. W ...[more]

PMID: 26436690

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SERRATE interacts with the nuclear exosome targeting complex to degrade primary miRNA precursors in Arabidopsis

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2020-04-15 | PXD014447 | Pride

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