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Fig. 5. The specific tetraspanin density distribution on a sucrose gradient is dependent on actin polymerization state. 2x10⁷ Jurkat cells were lysed at 4°C in MNE supplemented with 0.5% Triton X-100, and centrifuged overnight at 4°C in a three-step (42.5%, 30%, 5%) sucrose gradient, and 12 fractions of 280 ml were collected from the top. Proteins resolved by SDS-PAGE were analyzed by immunoblots with indicated mAbs. (A) Before lysis, cells were treated for 2 hours with 2 µM latrunculin (a depolymerizing agent), and an equal amount of the pool of fractions (1-4) or (5-8) is shown. CD71* represent an overexpressed immunoblot of CD71. Numbers under each plots represent densitometric values normalized to the untreated pool of fractions 5-8, except the LAT blot, which is normalized to the untreated pool of fractions 1-4. (B) Fifteen minutes before lysis, cells were electroporated with or without phalloidin, lanes 1-6 were loaded with 30 µl of each fraction, lanes 7-12 with only 6 µl.

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