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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Nat Cell Biol. 2011 May 22;13(6):715–721. doi: 10.1038/ncb2252

Figure 2. Knockdown of retromer by RNAi inhibits β2AR recycling and misroutes internalized β2ARs to lysosomes.

Figure 2

(a) Representative images from a visual assay for β2AR trafficking are shown. Stably transfected HEK 293 cells expressing FLAG-β2AR were transfected with either control siRNA or siRNA targeting the retromer component VPS35. In the “Agonist” condition, cells were incubated in the presence of the β2AR agonist isoproterenol (10 μM) and Alexa-conjugated M1 anti-FLAG for 25 min. In the “Agonist → Antagonist” condition, cells were incubated with isoproterenol for 25 min and then for an additional 45 min in the absence of isoproterenol (and in the presence of 10 μM of the β2AR antagonist alprenelol to prevent effects of any residual agonist). The scale bar represents 20 μm. (b) Flow cytometric analysis of β2AR recycling by uptake and efflux of bound M1 anti-FLAG antibody (n=4). (c) A time-course of recycling is shown for β2AR. The experiment was performed as in (b) but the duration after agonist washout was varied (n=4). (d) Representative confocal image from live cell imaging showing an endosome from a VPS35-1 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (e) A representative immunoblot assay of agonist induced FLAG-β2AR degradation. Detergent extracts were prepared from HEK 293 cells expressing FLAG-β2AR incubated in the absence of agonist or in the continuous presence of 10 μM isoproterenol for 4 h. Knockdown was verified by VPS35 immunoblot of the same lysates (middle panel), and equal loading was verified by immunoblotting for GAPDH (bottom panel). (f) FLAG-β2AR immunoblots were quantified by scanning densitometry across multiple experiments (n=3), and the percent receptor remaining after 4 h isoproterenol exposure was calculated. (g) Representative immunoblot showing isoproterenol induced β2AR degradation in cells depleted of VPS35 and treated with the vehicle dimethyl sulfoxide (DMSO), the lysosomal cathepsin inhibitor N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), or the proteosomal inhibitor epoxomicin (EPOX) (n=3). Data points are the mean ± SEM.