FIGURE 4.

Nedd4 is required for lysosomal targeting of the β₂AR. A, 293^β2AR cells were transfected with siRNA that target nothing, Nedd4, and Nedd4-2, stimulated for 6 h with 1 μm isoproterenol for the indicated times, fixed, permeabilized, and immunostained for the β₂AR (green) and LAMP2 (red). Colocalization is seen in the overlay panels (yellow). The data shown are from one of four independent experiments with identical results. B, whole cell lysates (20 μg) were probed with anti-WW2 domain antibody that recognizes both Nedd4 and Nedd4-2. The predicted molecular weight of human Nedd4 is 104,086 (NP_006145) and Nedd4-2 is 109,891 (NP_056092). C, 293^β2AR cells were transfected with siRNA targeting Mdm2 (top row) or AIP4 (bottom row), stimulated with 1 μm isoproterenol for indicated times and analyzed for receptor and LAMP2 distribution by immunostaining as described in A. The confocal panels displayed are representative of four independent experiments with identical results.