Figure 4. D₁R and D₅R association with AC isoforms.

A. Co-localization of hD₁R or hD₅R with AC5/6 and cAMP in HEK-hD₁R and HEK-D₅R cells. HEK-hD₁R and HEK-hD₅R cells, grown on coverslips, were treated with vehicle (Control) or fenoldopam (Fen, 1μmol/L) for 15 or 60 min. The cells were then washed, fixed, and triple immunofluorescence staining was performed. Fluorescence images were obtained using laser confocal scanning microscopy (Olympus Fluoview FV600) at excitation and emission wavelengths of 495/519, 579/603 and 632/648 nm. Myc-tagged D₁R or V5-tagged D₅R, green; AC5/6, blue; cAMP, red; Scale bar=20 μm.

B-C. The interaction of D₁R and D₅R with AC5/6 or with AC3

HEK-hD₁R and HEK-hD₅R cells were treated for 15 min at 37°C with vehicle (Control, C), fenoldopam (Fen, 1μmol/L), SCH23390 (S) (5 μmol/L), or combination of Fen and SCH23390 (F+S). Cell lysates were immunoprecipitated with specific polyclonal anti-AC5/6 antibody (Figure 4B) or anti-AC3 antibody (Figure 4C). Proteins were eluted from the immunocomplexes and probed with anti-Myc- (D₁R) (Figures 4B and 4C) or anti-V5- (D₅R) (Figure 4B) tagged antibodies. The immunoreactive bands were semi-quantified. Values are Mean ± SEM (n=4-6/group: ^*P<0.006 vs. all others, one-way factorial ANOVA, Tukey post-hoc test. One immunoblot is shown.

D. The interaction of D₁R or D₅R with AC5/6 in hRPT cells. hRPT cells were treated for 15 min at 37°C with vehicle (Control, C), fenoldopam (F, 1μmol/L), SCH23390 (S) (5umol/L), or combination (F+S) as in Figures 4B and 4C. Cell lysates were immunoprecipitated with polyclonal anti-AC5/6 antibody and proteins from immunocomplexes were probed with polyclonal rabbit anti-D₁R and -D₅R antibodies. The immunoreactive bands were semi-quantified. Values are Mean+SEM (n=6/group). ^*P<0.05, vs. all others, in AC5/6-D1R or AC5/6-D₅R, one-way factorial ANOVA, Tukey post-hoc test. One immunoblot for D₁R or D₅R is shown.