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Fig. 1. Gene expression of MAL, MAL2 and BENE in different cell lines and raft-association of these proteins in PC-3 cells. (A) Total RNA from the indicated cell lines was hybridized to cDNA probes specific to MAL, MAL2, BENE and {alpha}-actin as indicated. (B) Cells were extracted with 1% Triton X-100 at 4°C and subjected to centrifugation to equilibrium in sucrose density gradients. Aliquots from each fraction were analyzed by immunoblotting with antibodies specific to MAL, BENE and MAL2. Antibodies to cav-1 (a protein normally associated to rafts) and to calnexin (an ER-associated protein) were used as controls for the fractionation procedure. Two isoforms of cav-1, {alpha} and ß, are recognized by the anti-cav-1 antibody. The smear of bands between 30 and 40 kDa observed for MAL2 (*) represents glycosylated forms of this protein. Fractions 1-4 represent the 40% sucrose layer and contain the bulk of cellular membranes and cytosolic proteins and fractions 5-12 represent the 5-30% sucrose layer and contain rafts. The position of molecular weight markers in kDa is indicated on the right. (C) PC-3 cells stably expressing MAL-GFP or BENE-myc were extracted with 1% Triton X-100 at 4°C and subjected to centrifugation to equilibrium in sucrose density gradients. Aliquots from each fraction were analyzed by immunoblotting with anti-tag antibodies.





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