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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Dopamine-2 receptor extracellular N-terminus regulates receptor surface availability and is the target of human pathogenic antibodies from children with movement and psychiatric disorders

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

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