Figure 4. Active immunization selectively prevents tPA/GluN1 interaction and tPA-induced enhancement of NMDAR signaling without altering the basal Ca2+ conductivity. A, Schematic representation of NMDAR composed of GluN1/GluN2D subunits, including binding sites of αATD–GluN1, αCTD–GluN1, and αCTD–GluN2D antibodies, UBP145, and tPA. B, ATD–GluN1 immunized mice display antibodies specifically targeting the GluN1 subunit of NMDAR. Proteins extracts from naive mouse brain (n = 3) were subjected to immunoblots revealed with IgGs purified from either control mice (control IgGs) or ATD–GluN1 (120 kDa) immunized mice (αATD–GluN1). Parallel immunoblottings were performed and revealed with antibodies raised against either CTD–GluN1 (named αCTD–GluN1), known to reveal a band at ∼120 kDa, or CTD–GluN2D (named αCTD–GluN2D), known to reveal a band at ∼165 kDa. C, After immunization, mice display circulating antibodies against GluN1, capable of preventing the potentiating effect of tPA on GluN1/GluN2D subunit-containing NMDARs. NMDA induces Ca2+ influx in cortical neurons as measured by fura-2 video microscopy (N = 3, n = 150 cells). Coapplication of tPA (20 μg/ml; 45 min) potentiates the NMDA-evoked Ca2+ influx by 47% (N = 3, n = 108 cells). Neither UBP145 alone (0.2 μm; N = 3, n = 150 cells) nor αATD–GluN1 antibodies alone (0.01 mg/ml; N = 3, n = 108 cells) alter NMDA-induced Ca2+ influx. Both UBP145 (0.2 μm) and αATD–GluN1 (0.01 mg/ml) are capable of blocking this potentiating effect of tPA (N = 3, n = 150 cells and N = 3, n = 108 cells, respectively). Ctrl, Control; HBBSS, serum-free medium. Paired Student's t test (before vs after treatment), *p < 0.001. Vertical bars indicate SD.