Fig. 5

Human antibodies bind to mouse D2R extracellular N-terminus with a lower immunoreactivity. a Amino acid sequences of extracellular N-terminus of mouse D2R (D26E/A29P) and human WT D2R. Green = putative N-glycosylation sites; Red = amino acid change between species. b Schematic of D26E/A29P at cell surface. Red = 3xHA tag; Green = N-glycosylation site; Blue = D2R sequence; Purple = point mutation. c Confocal images after live immunolabeling of D26E/A29P-transfected HEK293 cells using an anti-HA antibody showed high levels of cell surface expression (scale bar = 50 μm). d Similar cell surface expression between WT D2R and D26E/A29P constructs was also confirmed by flow cytometry on live cells. No D2R expression from empty vector was observed. Representative data out of three independent experiments is shown. e Sera from anti-D2R antibody-positive movement and psychiatric disorders (MPD, n = 25) were incubated with empty vector-, WT D2R -, and D26E/A29P-transfected live HEK293 cells at 1:50 dilution, followed by AF647-conjugated anti-human IgG secondary antibody, and flow cytometry analysis. Percentage of sera binding to D26E/A29P (MFI %) was calculated using the formula described in Materials and methods. 60% patients (15/25) recognized D26E/A29P, whereas 40% (10/25) showed no immunoreactivity to D26E/A29P. Binding threshold is represented by solid line on graph. Representative data out of three experiments is shown. f Patient sera stained live murine hippocampal neurons, but no difference was observed between high or low serum binders to D26E/A29P mutant. g Patient sera did not stain live human neural stem cell-derived neurons, probably because these neurons did not express D2R on cell surface when immunolabeled with commercial anti-D2R antibody (scale bar = 25 μm)