FIG. 10.

Endogenous PDE4B2 is found within lipid rafts of human peripheral blood T-cell blasts. (A) Localization of endogenous PDE4B2 in resting T-cell blasts was assessed by confocal microscopy. Lipid rafts were identified by using fluorescein isothiocyanate-labeled cholera toxin, which binds to GM-1 (left panel). The PDE4B2 distribution (center panel) was determined by using a polyclonal anti-PDE4B2 rabbit serum and an Alexa Fluor 633-labeled secondary antibody. The GM-1-PDE4B2 overlay is shown in the right panel. T cells (10⁶) attached to a poly-l-lysine matrix were examined with a Zeiss LSM510 confocal microscope (63× objective). (B) Endogenous PDE4B2 is detected in the lipid raft compartment of T-cell blasts. Lysates from 250 × 10⁶ T cells were fractionated by sucrose gradient centrifugation to isolate the lipid raft fraction. The pelleted rafts and the soluble fraction were immunoblotted for PDE4B2 (upper panel), for total ERK (middle panel), or for GM-1 (lower panel).