Fig. 2: FCS data reveal β₂-AR diffusion on two different time scales.

a FCS curve derived from the basal membrane of a CHO-K1 cell transiently transfected with N-terminal-EGFP-β₂-AR (NT). The curve was fitted with the model 2D3DT (Eq. (4)) containing two diffusion times (t_D1  = 68.7 ± 1.13 ms, white and t_D2 = 0.75 ± 0.10 ms, light gray) and a relaxation time (t_T = 5.00 ± 5.68 µs, gray). The weighted residuals are shown on top. Fit results are mean ± s.e.m. b Diffusion constants, D_fast (light gray area) and D_slow, calculated from the corresponding diffusion times t_D1 and t_D2, respectively (Eq. (5)) of NT (light green, 6.08 ± 2.80 and 0.10 ± 0.02 µm² s⁻¹ for n = 12), IL3 (green, 8.09 ± 3.65 and 0.13 ± 0.06 µm² s⁻¹ for n = 12), S (meadow green, 11.0 ± 13.2 and 0.06 ± 0.03 µm² s⁻¹ for n = 10) and TAG (dark green, 4.74 ± 1.36 and 0.06 ± 0.01 µm² s⁻¹ for n = 6). c Fraction of molecules exhibiting fast diffusion for the same constructs as in (b). NT = 0.33 ± 0.05; IL3 = 0.31 ± 0.04; S = 0.07 ± 0.03 and TAG = 0.27 ± 0.10. Results for α_2A-AR are shown in Supplementary Fig. 5. Data are mean ± s.d. d Fraction of molecules exhibiting fast diffusion for NT at different positions on and partly on the plasma membrane. The lower cartoon represents the respective focal positions. The focal volume and the dimension of the cell are drawn to scale. The dimensions of the focal volume were 2.72 µm × 0.46 µm and the dimensions of the representative cell were 9 µm × 40 µm. Basal 0.22 ± 0.06; Basal + Cytosolic 0.44 ± 0.10; Apical + Cytosolic 0.42 ± 0.06; Apical 0.21 ± 0.03. Data are mean ± s.d. Diffusion constants for all the cell are shown in Supplementary Fig. 9. e Western Blot of untransfected (−) CHO-K1 and NT transfected (+) HEK293T and CHO-K1 cells. For both samples, the whole cell lysate (W), the separated cytosol (C) and membrane (M) fractions were loaded (see the section “Methods” for details). As a positive control for NT, HEK293T transfected with untagged β₂-AR (“β₂-AR”), and for EGFP, purified EGFP (“EGFP”) were used. As loading control the same gels were probed for Gβ after stripping. The full blots along with the controls can be seen in Supplementary Fig. 10. Blots for S construct can be seen in Supplementary Fig. 11.