Fig 1.
Characteristics of MasR antibodies used in the study.
pAb polyclonal antibody, H human, M mouse, R rat, C canine, B bovine, WB western blot, IHC immunohistochemistry, ICC immunocytochemistry, IF immunofluorescence, IP immunoprecipitation, IFC indirect flow cytometry, ELISA enzyme-linked immunosorbent assay.
Fig 2.
Detection of MasR mRNA in heart and kidney from wild type and MasR-KO mice.
Representative agarose gel (n = 3–5) showing reverse transcription polymerase chain reaction (RT-PCR) product obtained from hearts (A) and kidneys (B) of wild type (WT) and MasR-KO mice. MasR mRNA was undetectable in heart and kidney of MasR-KO mice. 18S ribosomal RNA (18S rRNA) was used as housekeeping gene.
Fig 3.
Western blot analysis of heart and kidney homogenates from wild type (WT) and MasR-KO mice.
The predicted molecular weight of MasR is about 37 kDa. As observed, the assayed antibodies generated different immunoreactivity patterns. In both cases there were no differences in the band patterns obtained with tissues from WT and MAS-KO mice. The membranes are representative of n = 4.
Fig 4.
Immunohistochemistry in heart and kidney from wild-type (WT) and MasR-KO mice.
Negative controls performed by omitting the primary antibody demonstrated minimal background immunostaining in heart (A) and kidney (B) sections. (A) In heart sections, NLS-1531 antibody stained predominantly the cytoplasm of cardiomyocytes with similar intensity in WT and MasR-KO hearts. The AAR-013 antibody stained the cardiomyocytes nucleus and weaker staining was observed in their cytoplasm. The same pattern was found in heart sections of MasR-KO mice. (B) In kidney sections, for NLS-1531 antibody, staining was mostly restricted to cytoplasm of numerous tubules cells with similar intensity in WT and MasR-KO kidneys. Antibody AAR-013 stained most intensely tubules cells nucleus and weaker staining was observed in their cytoplasm. Glomeruli were also stained positively. The same pattern was found in kidney sections of MasR-KO. Preincubation of the AAR-013 antibody with the blocking peptide provided by the vendors eliminated the immunohistochemical nuclear staining both in WT and MasR-KO mice kidney sections. Images are representative of n = 3. Bar, 50 μm.
Fig 5.
Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag antibody.
Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. The intense immunostaining obtained with the anti c-myc tag antibody indicated strong expression of MasR in HEK293T cells after transfection with the pcDNA 3.1/c-myc-MasR construct. A significant amount of the total MasR pool was present at the plasma membrane. No c-myc staining was observed in cells transfected with the empty vector pcDNA 3.1. Images are representative of 3 independent experiments.
Fig 6.
Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using MasR antibodies.
Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. In cells transfected with the pcDNA 3.1/c-myc-MasR construct, antibodies NLS-1531 and sc-54682 generated a similar staining pattern to that generated with the anti c-myc antibody. No staining was observed with any of these antibodies in cells transfected with the empty vector pcDNA 3.1. Antibody sc-135063 was capable of staining the plasma membrane of cells overexpressing the MasR but also generated widespread signals in cells transfected with the empty vector. For antibody AAR-013 there was no staining associated with the cell membrane. Intense immunocytochemical staining of identical distribution and intensity was revealed in cells transfected with the empty vector and those transfected with the c-myc-MasR construct. Images are representative of 3 independent experiments.
Fig 7.
Western blotting of HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag and three MasR antibodies.
Cells were transfected with varying amounts of a plasmid encoding the c-myc-tagged MasR (0.2 and 0.5 μg of pcDNA 3.1/c-myc-MasR).The anti-c-myc antibody detected a band of 37 kDa whose intensity increased as higher amount of DNA was transfected (arrow). Additionally, a ladder of higher molecular weight (MW) immunoreactive bands could be observed (asterisk). sc-54682 antibody reproduced the band pattern obtained with the c-myc antibody (arrow and asterisk). sc-135063 antibody revealed multiple bands close to 37 kDa with one of them of increasing intensity as higher amount of DNA was transfected (arrow) but did not produce a ladder of higher MW immunoreactive bands. High unspecific staining was attained with sc-135063 antibody. AAR-013 antibody revealed a pattern of immunoreactivity not comparable to those obtained with the other antibodies. Detection of β-tubulin was used as a protein loading control. The membranes are representative of 3 independent experiments.