Following 1 h blocking with 5% nonfat dry milk in phosphate buffered saline (PBS) containing 0.2% Tween 20 (PBS-T), the membrane was probed with antibody against Mas (1:1000) [2] and [20] during 2 h at room temperature. The membranes were washed 4 times for 15 min in PBS-T and incubated with anti-mouse

IgG-HRP-conjugated secondary antibody (1:2000) for 1 h. Afterward, the membranes were washed 4 times for 15 min in PBS-T, incubated with chemiluminescent agent (ECL plus, Amersham Biotechnology) for 1 min and exposed to a film to visualize protein bands. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000, Santa Cruz Biotechnology) bands were analyzed in parallel and used as a loading control for normalization of the Mas protein levels using the software ImageQuant™. Mas polyclonal antibody was produced in Mas knockout mice using as antigen MLN0128 nmr a 12 amino acid peptide (LAEEKAMNTSSR) corresponding to the NH2-terminal domain of the mouse Mas protein. This sequence has 100% homology with mouse and 91.6% homology with rat Mas and it is not present in any other known protein (see Target Selective Inhibitor Library clinical trial Fasta protein database, www.ebi.ac.uk/fasta33). To confirm our findings we repeated some immunoblotting experiments

with a commercial anti-Mas antibody (1:1000, Alomone). Cardiomyocytes were fixed in 2% paraformaldehyde solution diluted in PBS for 15 min. For immunostaining, cells were incubated with 5% bovine serum albumin (BSA) in PBS containing 5 mg/ml of saponin for 1 h followed by incubation with a polyclonal antibody against Mas raised in Mas deficient mice and diluted at 1:25 [2] and [20]. In order to confirm that the entry of the antibody into the cell was achieved, cardiac cells were probed with an antibody against the intracellular Ca2+ channel, the

type 2 ryanodine receptor (RyR2) (diluted 1:50, Affinity BioReagents) overnight Vorinostat research buy at 4 °C. Afterward, they were incubated with goat anti-mouse IgG conjugated with Alexa 633 for 1 h at room temperature. Each step was followed by washing the cells with PBS. The cells were mounted and viewed with a laser scanning confocal microscope (Zeiss 510 Meta-CEMEL ICB, UFMG). All confocal settings (aperture, gain and laser power) were determined at the beginning of the imaging session and these parameters were not changed. All data are expressed as mean ± SEM. Statistical significance was estimated using Student t-test (GraphPad Prism 4.0). The level of significance was set at p < 0.05. To evaluate the expression and localization of Mas in isolated ventricular myocytes from adult rats, we used western blotting and immunofluorescence-labeling techniques. As expected, it was observed that Mas is expressed in ventricular myocytes (Fig. 1A). Testicular samples were used as positive controls. Furthermore, this receptor was mainly localized in the sarcolemma of cardiomyocytes and absent in T-tubules (Fig. 1B).