Figure 6.

β₂AR induces GRK5 translocation to the cytoplasm for subsequent MR phosphorylation. (A) H9c2 cardiomyocytes were untreated or treated with 100 nM Aldo for 30 min. Subsequently, whole cell protein lysates were prepared and subjected to subcellular fractionation. Cytosolic and nuclear extracts fractions were separately immunoprecipitated (IP) with an anti-MR antibody, and the IPs were then immunoblotted (IB) for GRK5. Representative blots from six independent experiments are shown, including blots for MR to confirm equal amounts immunoprecipitated and for tubulin (cytosolic marker) and lamin A (nuclear marker) to confirm the identity of the lysate fraction (cytoplasm or nucleus, respectively). No GRK5 immunoreactivity could be detected in the MR IPs derived from the nuclear lysates. (B,C) H9c2 cardiomyocytes were treated with 10 µM salbutamol (Salb) for 30 min in the presence or absence of 10 µM U73122, and then, whole cell lysates were prepared to IP the MR, followed by IB for GRK5. Representative blots are shown in (B), including blots for MR to confirm equal amounts immunoprecipitated, and the relative densitometric quantitation of six independent experiments done in duplicate is shown in (C). * p < 0.05; n = 6.