Fig. 6. Tetraspanin engagement modifies the distribution of CD82 in sucrose density gradient. 2.107 Jurkat cells were cultured for 15 minutes at 37°C on plates coated, with unspecific antibodies or antibodies against CD9, CD53, CD63, CD81, or CD82. The cells were lysed 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, 12 fractions of 280 µl were collected from the top, and the insoluble pellet was recovered by addition of 50 µl SDS sample buffer. (A) CD82 immunoblot of the 12 fractions recovered from unstimulated (top) or CD82-stimulated (bottom) Jurkat cells. (B) Four sequential fractions were pooled (1-4 and 5-8), resolved together with the insoluble pellet by SDS-PAGE and immunoblotted with mAbs against CD82, CD81 or CD9. Stimulation conditions are indicated at the top.