Figure 12.

PFK-B does not target to lipid rafts/caveolae-enriched fractions. To separate caveolar microdomains from the other membranous and cytosolic components, an established sucrose fractionation system was used. Cos-7 cells were transiently transfected with the cDNA encoding V5-tagged PFK-B, 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 centrifugation. Twelve fractions were collected and equal amounts of protein from each fraction were separated by SDS-PAGE and blotted onto nitrocellulose. The distribution of PFK-B and Cav-3 were analyzed by immunoblotting with V5 and Cav-3 antibodies, respectively. Note that when expressed alone, PFK-B is detected at the bottom of the gradient (fractions 9 to 12; top). Similarly, in the presence of Cav-3, PFK-B does not co-fractionate with Cav-3 (detected mainly in fractions 4 and 5), which marks the position of caveolar microdomains) (bottom).