Figure 7. Dimerization of β1AR and RAGE in HEK293 cells coexpressing β1AR and RAGE.

(A) Coimmunoprecipitation of β1AR with RAGE to indicate the interaction between these two receptors. HEK293 cells were transfected with either Flag-RAGE or HA-β1AR or both. Forty-eight hours after transfection, total cellular proteins were used for immunoprecipitation assay with anti-HA (for β1AR) (1:1,000) overnight. The precipitation was followed by Western blotting with anti-Flag (for RAGE) (1:1,000). The cellular lysate protein (30 µg) was used as a positive control for Flag-RAGE expression and anti-HA for loading control. The experiments were repeated 3 times. (B) FRET intensities in HEK293 cells’ coexpression of either HS-β1AR or FS-β1AR with FC-RAGE were expressed as the interaction of proteins between Snap and Clip. ^†P < 0.05 versus HS-β1AR or FC-RAGE; **P < 0.01 versus FS-β1AR or FC-RAGE; 1-way ANOVA. (C) Cell surface expression of β1AR and RAGE detected by Snap substrate (top panel) or Clip (bottom panel) fluorescence. **P < 0.05 versus Mock; 1-way ANOVA. The averaged data were expressed as mean ± SEM from 3 individual experiments with triplicates. β1AR, β1-adrenergic receptor; RAGE, receptor for advanced glycation end-products; HEK, human embryonic kidney; FRET, fluorescence resonance energy transfer; HS-β1AR, HA-SNAP-β1AR; FS-β1AR, Flag-SNAP-β1AR; FC-RAGE, Flag-Clip-RAGE; Tb, benzyl-guanines-Lumi4Tb; c.p.s., counts per second; Fluo, benzylcytosine-Fluorescein.