(A) AT₁R/D₅R HEK 293 cells were treated with vehicle or Fen (1 μM for 5 min). Shown are FLIM images, histograms, and decay graphs of cells treated in the absence (top) or presence (middle and bottom) of acceptor (Ub). The left shift (arrow) of AT₁R lifetime in Fen-treated samples demonstrated the occurrence of FRET. The lifetime of AT₁R (donor) in the absence of acceptor was 2.32 ± 0.2 ns; it was 2.33 ± 0.2 ns in the presence of acceptor with vehicle treatment, indicating no energy transfer. With Fen treatment in the presence of acceptor, the donor lifetime had 2 peaks: the first was quenched at 1.68 ± 0.2 ns (τ₁) because of the occurrence of FRET, with a corresponding energy transfer efficiency of 27.6% ± 3.7%; the second was 2.24 ± 0.3 ns (τ₂), which represented the unquenched AT₁R. The FLIM patterns observed in the basal state and after Fen were similar in 4 different experiments. Quantification was performed in 16–30 cells from 1 of the 4 experiments. Data are mean ± SEM. (B) Human RPT cells were treated with vehicle, Fen (1 μM for 5 min), tunicamycin (Tunica; 10 μg/ml for 12 h), or tunicamycin plus Fen. Cell membrane fractions isolated by biotinylation were immunoprecipitated with normal mouse Ig M/G (lane 1), normal rabbit IgG (lane 2), anti-AT₁R mAb (lane 3), or anti-D₅R antibody (lane 4) and immunoblotted with anti-Ub (clone FK1) or anti-AT₁R mAb. (Ub)n, polyubiquitin chain; HC, heavy chain.