FIGURE 5:

Autoantibodies (AAb) alter membrane localization of β-arrestin. (A) HEK-β1AR cells transfected with plasmids containing the β-arrestin2-GFP fusion gene were plated on poly-l-lysine–coated coverslips, serum starved, pretreated with no IgG or affinity-purified IgG3(−) or IgG3(+) autoantibody, stimulated with Dob, fixed with 4% paraformaldehyde for 30 min, and mounted using prolong gold mountant with 4’,6-diamidino-2-phenylIndole (DAPI). The β-arrestin recruitment was assessed using confocal microscopy. Representative images of β-arrestin recruitment depicted by green fluorescence (bar = 10 μm). (B) Representative depiction of plot profile measurement of GFP fluorescence showing changes in fluorescence intensities from one edge to the other edge of the cell, which depicts the reduction of fluorescene intensity in the cytoplasm and increase at the plasma membrane following dobutamine (Dob treatment. (C) The relative ratio of plasma membrane (Mem) to cytoplasm (Cyt) intensity with Dob treatment in the presence of no IgG or IgG3(−) or IgG3(+) AAb (n = 3). * p<0.05 IgG3(+) vs. IgG3(−) AAb or no IgG. (D) Average changes (i.e,. clearance/loss) in cytoplasmic fluorescence intensity calculated over total fluorescence intensity of the cell reflecting β-arrestin recruitment, represented as percentage (∼ 30 cells/experiment [n = 3]). * p < 0.05 IgG3(+) AAb vs. no IgG or IgG3(−) AAb. (E) Percentage of cells showing effective β-arrestin recruitment following Dob in the presence of no IgG or IgG3(−) or IgG3(+) AAb (∼30 cells/experiment [n = 3]). *p < 0.05 IgG3(+) AAb vs. no IgG or IgG3(−) AAb.