Assessment of the (Pro)renin Receptor Protein Expression in Organs

Figure 1. Specificity validation of anti-N-(P)RR and ani-C-(P)RR antibodies. (A) The diagram of f(P)RR with different domains and the antigen sequences of anti-N-(P)RR and anti-C-(P)RR antibodies. TM: transmembrane domain, CP: cytoplasmic domain, a.a.: amino acid. (B,C) Cells were transfected with empty vector (EV) or (P)RR wild-type (WT)-overexpressed plasmid for 48 h and then the cell lysate and cell-culturing conditional medium (CM) were collected to examine the expression or secretion levels of different molecular forms of (P)RR by using the Western blot with anti-N-(P)RR (B) and anti-C-(P)RR antibodies (C). The experiments were repeated independently at least three times.

Figure 2. Different localization of f(P)RR, s(P)RR, and M8.9 fragment in the kidney. The kidney was collected from the SD rats and the exact localization of f(P)RR, s(P)RR, and M8.9 fragment in the kidney was examined by using immunohistochemical staining with anti-N-(P)RR (A, green) and anti-C-(P)RR antibodies (B, red). (C) The cell nucleus was stained by DAPI reagent (blue). (D) The merge image indicates the distribution and overlapping of different molecular forms of (P)RR and nuclei. Scale bar: 75 mm.

Figure 3. Different localization of f(P)RR, s(P)RR, and M8.9 fragment in the liver. The liver was collected from the SD rats and the exact localization of f(P)RR, s(P)RR, and M8.9 fragment in the liver was examined by using immunohistochemical staining with anti-N-(P)RR (A, green) and anti-C-(P)RR antibodies (B, red). (C) The cell nucleus was stained by DAPI reagent (blue). (D) The merge image indicates the distribution and overlapping of different molecular forms of (P)RR and nuclei. Scale bar: 75 mm.

Figure 4. Different localization of f(P)RR, s(P)RR, and M8.9 fragment in the pancreas. The pancreas was collected from the SD rats and the exact localization of f(P)RR, s(P)RR, and M8.9 fragment in the pancreas was examined by using immunohistochemical staining with anti-N-(P)RR (A, green) and anti-C-(P)RR antibodies (B, red). (E,F and I–L) The co-localization of f(P)RR, s(P)RR, and M8.9 fragment with insulin was examined by using immunohistochemical staining with anti-N-(P)RR (E, green), anti-C-(P)RR (I, green), and anti-insulin (F,J, red) antibodies. (C,G,K) The cell nucleus was stained by DAPI reagent (blue). (D,H,L) The merge image indicates the distribution and overlapping of different molecular forms of (P)RR, insulin, and nuclei. Star indicates vessel; dotted arrow indicates exocrine duct system; solid arrow indicates islets of Langerhans. Scale bar in (A–D): 75 mm. Scale bar in (E–L): 50 mm.

Figure 5. Different localization of f(P)RR, s(P)RR, and M8.9 fragment in the heart. The heart was collected from the SD rats and the exact localization of f(P)RR, s(P)RR, and M8.9 fragment in the heart was examined by using immunohistochemical staining with anti-N-(P)RR (A, green) and anti-C-(P)RR antibodies (B, red). (C) The cell nucleus was stained by DAPI reagent (blue). (D) The merge image indicates the distribution and overlapping of different molecular forms of (P)RR and nuclei. Solid arrow indicates endocardium. Scale bar: 50 mm.

Figure 6. Different localization of f(P)RR, s(P)RR, and M8.9 fragment in the brain. The brain was collected from the SD rats and the exact localization of f(P)RR, s(P)RR, and M8.9 fragment in brain cortex (A), third ventricle (B), and hippocampus (C) was examined by using immunohistochemical staining with anti-N-(P)RR (a in A–C and e in A–C, green) and anti-C-(P)RR antibodies (b in A–C and f in C, red). (A–C) The cell nucleus was stained by DAPI reagent (c in A–C and g in C, blue). (A–C) The merge image indicates the distribution and overlapping of different molecular forms of (P)RR and nuclei (d in A–C and h in C). Green arrow indicates (B) tanycytes and (C) radiatum layer and dentate gyrus. Scale bar is 75 mm in (A); 25 mm in (B); and 100 mm in (C).