Figure 4.
Overexpression of APP in HEK293 cells reduces α2AAR internalization. HEK293 cells were cotransfected with cDNA vectors encoding HA-α2AAR, together with APP or empty vector. A) Western blot image showing expression of exogenous APP in HEK293 cells, which have a very low level of endogenous APP. B) Internalization of cell surface HA-α2AAR was assessed by intact cell surface ELISA. Internalization is indicated by the decrease in the percentage of surface receptor (with t = 0 set as 100%). Data are means ± sem (n = 4 per condition). *P < 0.05 by Sidak’s multiple comparisons test; **P < 0.01 by 2-way ANOVA. C, D) Internalization of HA-α2AAR was examined by an Ab-prelabeling method. C) Representative images showing internalization of cell surface HA-α2AAR after 10 µM NE treatment (plus 1 µM prazosin and 1 µM propranolol). Arrows: perinuclear punctate staining, which is indicative of endocytosed receptors. Scale bars, 5 μm. D) Quantitation of α2AAR endocytosis. Data are means ± sem (n = 17–23 cells per condition). *P < 0.05, ***P < 0.001 (Student’s t test).