Figure 4.

Colocalization and coimmunoprecipitation of D₁R and IR in hRPTCs and in kidney sections. A and B, D₁R and IR colocalized (yellow, merge images) at the brush borders of RPTs in human (A) and rat (B) kidneys. C, D₁R colocalized (yellow, merge images) with IR at the perinuclear area in the basal state. Their colocalization increased with insulin treatment, reaching a maximum at 30 minutes. D, Alexa Fluor 488-labeled IR was used as the donor, whereas Alexa Fluor 555-labeled D₁R was used as the acceptor in the FRET dipole. The images (from left to right) are uFRET or uncorrected FRET, pFRET or processed or pure FRET, and E% or efficiency of energy transfer is around 30%. E, D₁R and IR coimmunoprecipitated and the amount of which was increased by insulin (Ins, 100nM/30 min) treatment (Tx). X, vehicle; Ab, antibody; IP, immunoprecipitation; Con, control. D₁R Ab was used for WB. The immunoblots to the right of the bar graphs indicate uniform expression of D₁R in the cells, with or without insulin treatment.