Fig. 4.

Altered protein composition in lipid rafts obtained from mantle cell plasma membranes. In A, Western blotting (W.B.) was used to confirm the distribution of flotillin 1 and Lyn in MCL P1 lipid rafts that had been purified by sucrose density gradient purification of Triton X-100 cell lysates as described under “Experimental Procedures.” The discontinuous sucrose gradient was fractionated as indicated, and fractions 1, 2, and 3 represent regions in the density gradient where lipid rafts should be found. This method was then used to purify lipid rafts from MCL cell lines and MCL patient cells, and the lipid rafts were separated by SDS-PAGE and analyzed by shotgun proteomics as described in Fig. 1. Selected proteins that are typical of lipid rafts are detailed in Table I. In B, Western blotting was used to compare the expression of raftlin, flotillin, Lyn, Cbp/PAG, 5-lipoxygenase, and actin in normal B cell (B1–B3) and MCL patient (P1–P5) whole cell lysates (Cells). Lipid rafts prepared from three patients and two MCL cell lines were also immunoblotted for these proteins. In C, RT-PCR was used to compare the mRNA expression of raftlin, 5-LO, and Cbp/PAG in normal B cells and cells derived from five MCL patients. Results are shown as mean ± S.E. (n = 5).