Fig. 2.

Effects of the X-link consisting of mouse GluN1 antibody and goat-anti-mouse IgG on NMDAR endocytosis and gating Blots shown in (a) are examples of Western analysis performed with antibodies against the GluN1 subunit (GluN1, upper blot) and actin (Actin, lower blot). The loaded cell lysates were prepared from cultured neurons treated without (“-”) or with (“+”) high NMDA/glycine (N + G: 1 mM NMDA and 100 μM glycine) in the absence (“-”) or presence (“+”) of X-link consisting of 5 μg/ml goat-anti-mouse IgG and 0.5, 2.5 or 5 μg/ml GluN1 antibody, as indicated above the blots. Immediately after the treatment, the cell membrane impermeable crosslinker, BS³ [Bis(sulfosuccinimidyl) suberate] , was applied to neurons at 4 °C as indicated with “+” below the blots. The ratio of the band intensity showing intracellular GluN1 subunit protein versus that of actin protein was calculated and normalized to the ratio in neurons without any treatment except BS³ (=100 %, control, dashed line in bar graphs). Bar graphs show summary data (mean ± SEM). ##: P < 0.01 (independent t-test) in comparison with control. b Summary data showing single-channel activity of NMDARs evoked by NMDA (10 μM) and glycien (3 μM) added into recording pipettes with (filled bars) or without (open bars) inclusion of X-link consisting of 2.5 μg/ml GluN1 antibody and 5 μg/ml goat-anti-mouse IgG