Figure 5.

FDPS modulates β2AR internalization and down-regulation in a Rab5-dependent manner. A) Effect of ALN on β2AR internalization into early endosomes. 293β2AR cells were treated with 50 μM ALN for 48 h and then stimulated with 10 μM ISO for 30 min. Cells were then fixed, permeablized, and stained with primary antibodies (polyclonal anti-Flag antibody for Flag-β2AR and a monoclonal antibody for EEA1) and respective TRIC- or FITC-conjugated secondary antibodies. Images were visualized under confocal microscopy. B) Effect of ALN on β2AR internalization. 293β2AR were mock-treated or treated with ALN and stimulated with 10 μM ISO for 30 min to induce internalization. Flow cytometry was used to measure amount of β2AR at the cell surface. Percentages of β2AR remained at the cell surface on ISO stimulation were calculated relative to surface receptor level without ISO treatment. ***P < 0.001. C) Effect of ALN on β2AR trafficking into late endosomes. 293β2AR cells were treated as in A and then stimulated with 10 μM ISO for indicated time. Immunostaining was performed to detect Flag-β2AR and LAMP2. D) Effect of wild-type or mutant Rab5 expression on β2AR down-regulation. 293β2AR cells were transfected with wild-type or mutant (S34N) GFP-Rab5 expression plasmids and after 48 h, cells were treated with 10 μM ISO for 3 h. Cell extracts were subjected to immunoblotting to detect receptor levels. Anti-GFP antibody was used to detect the expression of GFP-Rab5 fusion protein. E) Effect of ALN on Rab5 membrane association. 293β2AR cells were treated with 50 μM ALN for 48 h followed by 10 μM ISO stimulation for 30 min. Cells were lysed, and membrane-associated fractions were isolated using the Mem-PER Mammalian Membrane Protein Extraction Kit (Pierce). Immunoblotting was done on the membrane-associated fraction (MAF) and the whole cell extract (WCE) using indicated antibodies. F) Effect of ALN on Rab5 localization. 293β2AR cells transfected with GFP-Rab5 were mock-treated or treated with ALN for 48 h and then subjected to 10 μM ISO stimulation (or control vehicle treatment) for 30 min. Cells were fixed, permeabilized, and stained with anti-Flag antibody. Images were visualized with a confocal fluorescence microscope. G) Effect of Rab5 overexpression on the inhibition by ALN on β2AR down-regulation. 293β2AR cells were transfected with Rab5 expression plasmid or control plasmid pCDNA3.1, and after 48 h cells were treated with 25 μM ALN or control vehicle for 24 h. Cell were then subjected to 10 μM ISO stimulation (or control vehicle treatment) for 2 h, and extracts were analyzed by immunoblotting.