Fig. 2: The two-step labeling of cell-surface AMPARs ectopically expressed in HEK293T cells.

a Western blotting analyses of HEK293T cells after the two-step labeling. HEK293T cells transfected with GluA2^flip(Q) (AMPAR(+)) or vector control (AMPAR(−)) were treated with 2 μM CAM2(TCO) for 4 h followed by the addition of 1 μM Tz(Fl) for 5 min, or treated with 2 μM CAM2(Fl) for 4 h in the presence or absence of 50 μM NBQX in culture medium at 37 °C. The cell lysates were analyzed by western blotting using anti-fluorescein or anti-GluA2/3 antibody. Quantification of the band intensity is shown in Supplementary Fig. 3. When CAM2(Fl) is added in serum free medium, strong bands around 70 kDa in lane #6 and #7 disappear (for details see ref. ²⁶). b Effects of PNGase F treatment on the western blotting of labeled AMPAR in HEK293T cells expressing GluA2. Lower image shows the overlay of anti-Fl image and anti-GluA2/3 image for lane #3 and #4. Two-step labeling was conducted as described in (a). PNGase F (1000 units/100 μL) was added to the cell lysate. For details, see Methods section. c Confocal live imaging of the HEK293T cells labeled with 2 μM CAM2(TCO) and 0.1 μM Tz(Ax488). Labeling was conducted as described in (a). mCherry-F was utilized as a transfection marker. Scale bars, 10 μm. d Reaction kinetics of tetrazine ligation on live cells by confocal live imaging of the HEK293T cells labeled with 2 μM CAM2(TCO) after addition of 0.3 μM Tz(Ax488) at 37 °C. In left, confocal images are shown. Scale bars, 20 μm. In right, time-course of the fluorescent intensity of Alexa 488 is shown (n = 6 cells). Data are represented as mean ± s.e.m.