Figure 3.

GRK5 phosphorylates and inhibits the MR in cardiac myocytes. (A) Western blotting for phosphoserine content of immunoprecipitated MR in response to 100 nM aldosterone (Aldo) stimulation for 15 min or vehicle (DMSO) in GRK5-overexpressing (GRK5-OE) or control, mock lentivirus-infected (EV) H9c2 myocyte lysates or in lysates from myocytes that had GRK5 genetically deleted (GRK5-KO) and their respective control cells (CNCV, CRISPR (Clustered regularly interspaced short palindromic repeats) negative control virus). IP: immunoprecipitation; IB: Immunoblotting. Representative blots are shown in (A) and the relative densitometric quantitation of five independent experiments performed in duplicate is shown in (B). * p < 0.05, vs. EV; ^# p < 0.05, vs. CNCV; n = 5. (C) Immunoblotting for GRK5 in extracts from H9c2 cardiomyocytes, transfected with empty vector/mock lentivirus (EV), full-length wild-type GRK5-encoding lentivirus to overexpress GRK5 (OE), CRISPR negative control (mock) lentivirus (CNCV), or CRISPR rat GRK5-specific lentivirus to knockout GRK5 (KO). Total protein extracts were prepared 48 h post-infection and then separated on a 4%–20% SDS-PAGE gel. A representative blot is shown, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as loading control, of five independent experiments performed in duplicate, confirming GRK5 overexpression and deletion in OE and KO cells, respectively. (D) Transcriptional activity of the MR in response to various concentrations of Aldo in H9c2 cardiomyocytes overexpressing GRK5 (GRK5-OE) or having GRK5 genetically deleted (GRK5-KO). EV: Empty vector; CNCV: CRISPR negative control virus; * p < 0.05, between GRK5-KO and EV or CNCV; ** p < 0.05, between GRK5-OE and EV or CNCV; n = 5 independent measurements with triplicate samples per Aldo concentration.