Fig. 6: The two-step labeling of cell-surface NMDARs in HEK293T cells and neurons.
From: Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking
a Chemical structure of CNM(TCO) for two-step labeling reagent for NMDARs. Gly glycine. b Two-step labeling of cell-surface NMDARs ectopically expressed in HEK293T cells. In left, schematic illustration is shown. In right, western blotting analyses after the two-step labeling are shown. HEK293T cells transfected with NR1-1 and NR2A or vector control were treated with 10 μM CNM(TCO) for 4 h followed by the addition of 1 μM Tz(Fl) for 5 min in the presence or absence of 250 μM Pzf in culture medium at 37 °C. The cell lysates were analyzed by western blotting using anti-fluorescein or anti-NR2A antibody. c Determination of t1/2surface of NMDARs in HEK293T cells by western blotting. In left, representative results of western blotting are shown. In right, time-course of the labeled band is shown (n = 3 biological replicates). d Two-step labeling of cell-surface NMDARs endogenously expressed in cultured cortical neurons. In left, schematic illustration is shown. In right, western blotting analyses after the two-step labeling are shown. The cultured cortical neurons were treated with 10 μM CNM(TCO) for 10 h followed by the addition of 1 μM Tz(Fl) for 5 min in the presence or absence of 250 μM Pzf in culture medium at 37 °C. The cell lysates were analyzed by western blotting using anti-fluorescein or anti-NR2A antibody. e Determination of t1/2surface of NMDARs in neurons by western blotting. In left, representative results of western blotting are shown. In right, time–course of the labeled band is shown (n = 3 biological replicates). Data are represented as mean ± s.e.m.
