Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Role of GRK4 in the regulation of the renal ETB receptor in hypertension

doi: 10.1096/fj.201902552R

Figure Lengend Snippet: ETBR phosphorylation and GRK4 expression in renal cortex of WKY and SHRs. A, ETBR protein expression determined by immunoblotting. Results are expressed as the ratio of ETBR and GAPDH densities (N = 5/group, P = NS). B, Renal cortical ETBR phosphorylation determined by co-immunoprecipitation with immunoblotting. Renal cortical lysates were prepared (renal cortex of WKY rats for negative and positive control group), and mouse anti-phosphoserine antibody was used for immunoprecipitation, with normal mouse IgG as negative control, and mouse anti-ETBR antibody as positive control. The immune complexes were precipitated using agarose-A/G beads and immunoblotted for ETBR. NC indicates negative control and PC indicates positive control. *P < .05 vs WKY (N = 5/group). C and D, GRK4 mRNA and protein expressions in renal cortex of WKY and SHRs. *P < .05 vs WKY (N = 5/group). Data are expressed as Mean ± SE

Article Snippet: The immunoprecipitates were subjected to immunoblotting with rabbit anti-ETBR antibody (1:1000; Abcam) or anti-phosphoserine antibody.

Techniques: Expressing, Western Blot, Immunoprecipitation, Positive Control, Negative Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Role of GRK4 in the regulation of the renal ETB receptor in hypertension

doi: 10.1096/fj.201902552R

Figure Lengend Snippet: Effect of the intrarenal arterial infusion of the ETBR agonist BQ3020 on urine flow and sodium excretion in hGRK4γ WT and hGRK4γ 142V transgenic mice. A, Systolic, diastolic, and mean arterial blood pressures (SBP, DBP, and MBP, respectively), measured under pentobarbital anesthesia in hGRK4γ WT and hGRK4γ 142V transgenic mice, *P < .05 vs WT (N = 5/group). B and C, Basal urine flow (24 hours, V) and basal absolute sodium excretion (24 hours, UNaV). P = NS (N = 5/group). D and E, Urine flow (V) and absolute sodium excretion (UNaV). Varying doses (0.1, 0.5, 1.0 μg/kg/min) of ETBR agonist, BQ-3020 were infused at a rate of 0.04 mL/min for 40 minutes, preceded by a control period (40 minutes), and followed by a recovery period (40 minutes) in which the vehicle, normal saline, alone, was infused via the right supra-renal artery of anesthetized mice. #P < .05 vs control. *P < .05 vs WT (N = 5/group). F, Renal cortical ETBR phosphorylation determined by coimmunoprecipitation with immunoblotting. Renal cortical lysates were prepared (renal cortex of hGRK4γ 142V transgenic mice for negative and positive control group), and mouse anti-phosphoserine antibody was used for immunoprecipitation, with normal mouse IgG as negative control, and mouse anti-ETBR antibody as positive control. The immune complexes were precipitated using agarose-A/G beads and immunoblotted for ETBR. NC indicates negative control and PC indicates positive control. *P < .05 vs WT (N = 4/group). Data are expressed as Mean ± SE

Article Snippet: The immunoprecipitates were subjected to immunoblotting with rabbit anti-ETBR antibody (1:1000; Abcam) or anti-phosphoserine antibody.

Techniques: Transgenic Assay, Western Blot, Positive Control, Immunoprecipitation, Negative Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Role of GRK4 in the regulation of the renal ETB receptor in hypertension

doi: 10.1096/fj.201902552R

Figure Lengend Snippet: Effect of GRK4 silencing on ETBR function. A, Effect of ETBR BQ3020 on Na+-K+-ATPase activity in RPT cells from WKY and SHRs. The cells were incubated with BQ3020 (10−8 M) or vehicle (dH2O) for 30 minutes. Results are expressed as μmol phosphate released per mg protein per hour. #P < .05 vs WKY. *P < .05 vs control (N = 6/group). B, Effect of GRK4-specific siRNA on GRK4 expression in RPT cells from SHRs. GRK4-specific siRNA (5 nM) or nonsilencing “mock” siRNA (negative control) was transfected into RPT cells for 72 hours. Untransfected cells served as an additional negative control. The mRNA expression of GRK4 related to control (RPT cells) was quantified using quantitative real-time PCR. *P < .05 vs others (N = 4/group). C, Effect of the ETBR agonist BQ3020 on Na+-K+-ATPase activity in RPT cells from SHRs with or without GRK4 silencing with siRNA. The cells were incubated with BQ3020 (10−8 M) or vehicle (dH2O) for 30 minutes. Results are expressed as μmol phosphate released per mg protein per hour. *P < .05 vs control (N = 6/group). D, Effect of renal UTMD-mediated GRK4 siRNA delivery on renal cortical GRK4 protein expression in SHRs. *P < 0.05 vs control (N = 4/group). E and F: Urine flow (V) and absolute sodium excretion (UNaV) in SHRs after downregulation of renal GRK4 expression by UTMD. The SHRs were subjected to UTMD-targeted GRK4 siRNA delivery to the kidney, given every 3 days for a total of six treatments. After the UTMD treatment, varying doses (0.1, 0.5, 1.0 μg/kg/min) of the ETBR agonist BQ-3020 were infused at a rate of 0.04 mL/min for 40 minutes, preceded by a control period (40 minutes), and followed by a recovery period (40 minutes) in which the vehicle, normal saline, alone was infused via the right supra-renal artery of anesthetized mice. *P < .05 vs control (N = 6/group). G, Renal cortical ETBR phosphorylation determined by co-immunoprecipitation with immunoblotting. Renal cortical lysates were prepared (renal cortex of SHR rats for negative and positive control group), and mouse anti-phosphoserine antibody was used for immunoprecipitation, with normal mouse IgG as negative control, and mouse anti-ETBR antibody as positive control. The immune complexes were precipitated using agarose-A/G beads and immunoblotted for ETBR. NC indicates negative control and PC indicates positive control.*P < .05 vs control (N = 4/group). Data are expressed as Mean ± SE

Article Snippet: The immunoprecipitates were subjected to immunoblotting with rabbit anti-ETBR antibody (1:1000; Abcam) or anti-phosphoserine antibody.

Techniques: Activity Assay, Incubation, Expressing, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Positive Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Role of GRK4 in the regulation of the renal ETB receptor in hypertension

doi: 10.1096/fj.201902552R

Figure Lengend Snippet: Interaction between ETBR and GRK4 in WKY and SHR RPT cells. A, Colocalization of ETBR and GRK4 in RPT cells from WKY and SHRs. Colocalization appears as yellow after merging the images of Alexa Fluor 546-tagged ETBR (red) and Alexa Fluor 488-tagged GRK4 (green). B, Co-immunoprecipitation of GRK4 and ETBR in RPT cells from WKY and SHRs. The cell lysates (lysates of RPT cells from SHR for negative and positive control group) were immunoprecipitated with mouse anti-GRK4 antibody, with normal mouse IgG as negative control and mouse anti-ETBR antibody as positive control, and then, immunoblotted with ETBR antibody. NC indicates negative control and PC indicates positive control. *P < .05 vs WKY (N = 4/group). Data are expressed as Mean ± SE

Article Snippet: The cells were double-immunostained for ETBR and GRK4, using rabbit anti-ETBR antibody (1:100) and mouse anti-GRK4 antibody (1:100), respectively, and then, with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific), respectively.

Techniques: Immunoprecipitation, Positive Control, Negative Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Role of GRK4 in the regulation of the renal ETB receptor in hypertension

doi: 10.1096/fj.201902552R

Figure Lengend Snippet: ETBR phosphorylation and GRK4 expression in renal cortex of WKY and SHRs. A, ETBR protein expression determined by immunoblotting. Results are expressed as the ratio of ETBR and GAPDH densities (N = 5/group, P = NS). B, Renal cortical ETBR phosphorylation determined by co-immunoprecipitation with immunoblotting. Renal cortical lysates were prepared (renal cortex of WKY rats for negative and positive control group), and mouse anti-phosphoserine antibody was used for immunoprecipitation, with normal mouse IgG as negative control, and mouse anti-ETBR antibody as positive control. The immune complexes were precipitated using agarose-A/G beads and immunoblotted for ETBR. NC indicates negative control and PC indicates positive control. *P < .05 vs WKY (N = 5/group). C and D, GRK4 mRNA and protein expressions in renal cortex of WKY and SHRs. *P < .05 vs WKY (N = 5/group). Data are expressed as Mean ± SE

Article Snippet: The cells were double-immunostained for ETBR and GRK4, using rabbit anti-ETBR antibody (1:100) and mouse anti-GRK4 antibody (1:100), respectively, and then, with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific), respectively.

Techniques: Expressing, Western Blot, Immunoprecipitation, Positive Control, Negative Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Role of GRK4 in the regulation of the renal ETB receptor in hypertension

doi: 10.1096/fj.201902552R

Figure Lengend Snippet: Effect of GRK4 silencing on ETBR function. A, Effect of ETBR BQ3020 on Na+-K+-ATPase activity in RPT cells from WKY and SHRs. The cells were incubated with BQ3020 (10−8 M) or vehicle (dH2O) for 30 minutes. Results are expressed as μmol phosphate released per mg protein per hour. #P < .05 vs WKY. *P < .05 vs control (N = 6/group). B, Effect of GRK4-specific siRNA on GRK4 expression in RPT cells from SHRs. GRK4-specific siRNA (5 nM) or nonsilencing “mock” siRNA (negative control) was transfected into RPT cells for 72 hours. Untransfected cells served as an additional negative control. The mRNA expression of GRK4 related to control (RPT cells) was quantified using quantitative real-time PCR. *P < .05 vs others (N = 4/group). C, Effect of the ETBR agonist BQ3020 on Na+-K+-ATPase activity in RPT cells from SHRs with or without GRK4 silencing with siRNA. The cells were incubated with BQ3020 (10−8 M) or vehicle (dH2O) for 30 minutes. Results are expressed as μmol phosphate released per mg protein per hour. *P < .05 vs control (N = 6/group). D, Effect of renal UTMD-mediated GRK4 siRNA delivery on renal cortical GRK4 protein expression in SHRs. *P < 0.05 vs control (N = 4/group). E and F: Urine flow (V) and absolute sodium excretion (UNaV) in SHRs after downregulation of renal GRK4 expression by UTMD. The SHRs were subjected to UTMD-targeted GRK4 siRNA delivery to the kidney, given every 3 days for a total of six treatments. After the UTMD treatment, varying doses (0.1, 0.5, 1.0 μg/kg/min) of the ETBR agonist BQ-3020 were infused at a rate of 0.04 mL/min for 40 minutes, preceded by a control period (40 minutes), and followed by a recovery period (40 minutes) in which the vehicle, normal saline, alone was infused via the right supra-renal artery of anesthetized mice. *P < .05 vs control (N = 6/group). G, Renal cortical ETBR phosphorylation determined by co-immunoprecipitation with immunoblotting. Renal cortical lysates were prepared (renal cortex of SHR rats for negative and positive control group), and mouse anti-phosphoserine antibody was used for immunoprecipitation, with normal mouse IgG as negative control, and mouse anti-ETBR antibody as positive control. The immune complexes were precipitated using agarose-A/G beads and immunoblotted for ETBR. NC indicates negative control and PC indicates positive control.*P < .05 vs control (N = 4/group). Data are expressed as Mean ± SE

Article Snippet: The cells were double-immunostained for ETBR and GRK4, using rabbit anti-ETBR antibody (1:100) and mouse anti-GRK4 antibody (1:100), respectively, and then, with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific), respectively.

Techniques: Activity Assay, Incubation, Expressing, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Positive Control