Figure 3.

Recombinant expression of Cav-3 induces the targeting of PFK-M to caveolae-enriched membrane fractions. Caveolar microdomains were separated from the bulk of cellular membranes and cytosolic components using sucrose gradient fractionation. Cos-7 cells were transiently transfected with the cDNA encoding V5-tagged PFK-M, alone or in combination with Cav-3. Thirty-six hours after transfection, cells were lysed in a buffer containing 1% Triton X-100, and subjected to sucrose gradient ultracentrifugation. Twelve fractions were collected, and equal amounts of protein from each fraction (10 μg) were separated by SDS-PAGE and blotted onto nitrocellulose. The distribution of PFK-M and Cav-3 were analyzed by immunoblotting with V5 and Cav-3 antibodies, respectively. Note that when expressed alone, PFK-M is detected at the bottom of the gradient (A, fractions 8 and 12). In contrast, in the presence of Cav-3, PFK-M co-fractionates with Cav-3 (B, fractions 4 and 5), which marks the position of caveolar microdomains.