Flotillin 2

Cells or vesicles were homogenized in RIPA lysis buffer (150 mM NaCl, 0.1% SDS, 0.5% Na-deoxycholate, 1% Triton X-100, 50 mM Tris, pH 7.2). Up to 50 µg of protein were separated by SDS–PAGE (8% gels) and transferred onto a nitrocellulose membrane (75 min at 10 V). The blocked membrane was incubated with primary antibodies specific to EMMPRIN (#sc-13976), MUC-1 (#sc-7313), TSG101 (#sc-7964, all three from Santa Cruz), Flotillin-2 (#610383, BD), Tubulin (#05-829, Millipore), p38 MAPK total protein (#9212), phospho-p38 MAPK (#9211), c-jun total protein (#9165), and phospho-c-jun (#9261, all four from cell signaling). Signals were detected after incubation with anti-rabbit (#sc-2004) or anti-mouse (#sc-2005, both from Santa Cruz) HRP-labeled secondary antibodies using ECL^™ Prime (Amersham Biosciences). Ponceau S staining was routinely used as loading control. Zymography was performed as previously described (Hagemann et al., 2004 (link)). Briefly, cell lysates (35 µg), supernatants (12 µl), or MV (35 µg) were loaded onto SDS–PAGE gels (8%) supplemented with 1 mg/ml gelatine. After electrophoresis, gels were incubated overnight in renaturation buffer (200 mM NaCl, 5 mM CaCl₂, 0.02% Brij-35, pH 7.5) and stained with Coomassie brilliant blue.