Mouse anti grk4 antibody

The renal cortex or RPT cells were lysed, and then, equal amounts of lysates (300 μg protein/mL supernatant) were incubated with affinity-purified mouse anti-GRK4 antibody (5 μg/mL, GRK4/ETBR coimmunoprecipitation; Santa Cruz Biotechnology, Dallas, TX) or mouse anti-phosphoserine or mouse ETBR antibody (5 μg/mL, ETBR phosphorylation; Abcam, Cambridge, UK) for 1 hour and protein G plus-agarose overnight at 4°C. The immunoprecipitates were subjected to immunoblotting with rabbit anti-ETBR antibody (1:1000; Abcam) or anti-phosphoserine antibody. Anti-ETBR antibody or anti-phosphoserine antibody (positive control) and IgG (negative control) were used as the immunoprecipitants to evaluate the specificity of the bands found on the immunoblots.

Kidneys from WKY rats and SHRs were fixed with 4% of paraformaldehyde and dehydrated in increasing concentrations of ethanol. The samples were embedded in paraffin, sectioned into 4 μm-thick slices of kidneys, mounted on glass slides, and double-immunostained with rabbit anti-AT₂R antibody (1:100, Abcam, Cambridge, U.K.; ab254561) and mouse anti-GRK4 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, U.S.A.). Then, Alexa Fluor 546-conjugated goat anti-rabbit IgG secondary antibody (1:200, Thermo Fisher Scientific; A-11010) and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:200, Thermo Fisher Scientific; A-11001) were added and incubated for 1 h at room temperature. The nuclei in the kidneys were stained with DAPI and the kidney sections examined using laser scanning confocal microscope and the Olympus Fluoview FV300 version 3C Acquisition Software. Colocalization was quantified through Pearson’s correlation coefficient by Image Pro Plus software [31 (link),32 (link)].