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Fig. 3. Tetraspanin density distribution on a sucrose gradient depends on the presence of cholesterol but not of tetraspanin post-translational modifications. 2x10⁷ Jurkat cells were lysed at 4°C in MNE buffer supplemented with 0.5% Triton X-100. Lysates were centrifuged overnight at 4°C in a three-step (42.5%, 30%, 5%) sucrose gradient and 12 fractions of 280 µl were collected from the top. After non-reducing SDS-PAGE, immunoblotting was carried out with anti-CD82 (A, bottom, B) and anti-CD9 (A, top, C) antibodies. (A) Jurkat-cell lysis was performed in the presence or absence of 0.1% saponin, a specific cholesterol detergent, as indicated. (B) Before Jurkat-cell lysis, cells were treated overnight with the glycosylation inhibitor tunicamycin. (C) Daudi cells expressing wild-type CD9, or unpalmitoylated CD9 were analysed. Lanes were loaded with 30 µl (A, top, all lanes; B,C, lanes 1-6) or 6 µl (B,C, lanes 7-12) of each fraction.

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