ed as a percentage of the corresponding CTR worth (taken as 100%). Every of those assays was repeated utilizing various preparations (see “n” values inside the corresponding figure legends). The Scion Image application was utilised to quantify the band intensities. In preliminary experiments, the nitrocellulose membranes were stained with Ponceau Red to assess protein loading. Herrera et al. [38] demonstrated that several anti-Ang II receptors antibodies available from commercial sources display 
nonspecific binding in kidney and in other tissues. Inside the present operate, the specificities of your major antibodies against AT1R and AT2R were confirmed inside the present study by preadsorption experiments using a matrix of human Ang II type 2 receptor recombinant full-length protein (amino acids 163, ab157871, Abcam), as in [39] for the anti-renin antibody. A three-fold mass of the immunizing peptide was incubated with all the antibodies: (i) sc1173 Santa Cruz, anti-AT1R raised against a peptide mapping an N-terminal extracellular domain of AT1R (human); or (ii) sc9040 Santa Cruz, anti-AT2R raised against amino acids 22163 of AT2R (human). After centrifugation and dilution on the supernatants (1:500), immunostaining demonstrated the presence in the AT1R band in renal membranes utilizing the AT1R-containing solution, whereas no signal appeared when the AT2R-containing remedy was utilized (Figure 1). These antibodies against AT1R and AT2R were those utilised in the immunoassays described above.The activities of PKC and PKA were measured as in [8], [36].The activities of PKC and PKA linked with all the isolated membranes of renal and cardiac origin were measured by SDS-PAGE and imunoblotting for AT1R, AT2R, ERK1 and phospho-ERK1/2 had been carried out as in [8], [36] applying the specific antibodies. Briefly, ” the proteins of
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