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