Fig. 2.

Patient antibodies do not bind to the three extracellular loops of D2R or to D1R extracellular N-terminus. a, b Open reading construct of WT D2R, and ^NTermD1RχD2R constructs in which the extracellular N-terminus of D2R was swapped for the extracellular N-terminus of D1R. M = methionine; AA = amino acid; Red = 3x haemagglutinin (HA) tag; Blue = D2R sequence; Pink = D1R sequence; TM1 = first transmembrane domain; I1 = first intracellular loop; Green = N-glycosylation site. c Confocal images after live immunolabeling of WT D2R or ^NTermD1RχD2R-transfected HEK293 cells using an anti-HA antibody showed similar levels of cell surface expression for both constructs (scale bar = 50 μm). d Similar cell surface expression of WT D2R and ^NTermD1RχD2R 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 controls (CTL; n = 32) and movement and psychiatric disorders (MPD; n = 35) were incubated with empty vector- and WT D2R-transfected live HEK293 cells at 1:50 dilution, followed by AF647-conjugated anti-human IgG secondary antibody, and flow cytometry analysis. Anti-D2R antibody was detected in 35/35 MPD and 0/32 controls. f Sera from CTL (n = 32) and anti-D2R antibody-positive MPD (n = 35) were incubated with empty vector- and ^NTermD1RχD2R-transfected live HEK293 cells at 1:50 dilution, followed by AF647-conjugated anti-human IgG secondary antibody, and flow cytometry analysis. Antibodies against the three D2R extracellular loops or the D1R extracellular N-terminus were detected in 0/35 MPD and 0/32 controls. ∆ mean fluorescence intensity (∆MFI) was calculated using MFI obtained with empty vector- and WT D2R- and ^NTermD1RχD2R-transfected HEK293 cells. Positivity threshold was determined by ∆MFI of three SD above the mean of CTL (solid line on graph). Representative dot plot out of three experiments is shown