FIGURE 5.

Absence of βc receptor ubiquitination does not lead to increased recycling. A, WT βc and βc K16R-expressing HEK293 cell lines were treated with or without 40 μm monensin in suspension for 30 min. After cytospinning, cells were fixed and immunostained with anti-βc antibodies. Note the increase in intracellular βc vesicles seen in monensin-treated cells (left panels), which were not observed in cells expressing βc K16R receptors (right panels). For this assay, we used HEK293 cell in suspension because βc cell surface labeling was difficult to interpret in adherent monensin-treated HEK293 cells. B, same as in A except βc cell surface levels were measured by flow cytometry and are expressed as mean MFI ± S.E. minus isotype control MFI in the absence or presence of inhibitors; n = 3. WT βc MFI, 0 min (−Mon) = 7,686 ± 875; 0 min (+Mon) = 3,413 ± 334; 30 min (−Mon) = 7,713 ± 869; 30 min(+Mon) = 2,887 ± 316. βc K16R MFIs: 0 min (−Mon) = 22,767 ± 3252; 0 min (+Mon) = 22,622 ± 3576; 30 min (−Mon) = 23,171 ± 3630; 30 min (+Mon) = 22,556 ± 3,623.