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1.
Figure 9

Figure 9. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Expression and colocalization of renin and of the renin receptor in placenta. Sequential sections of placenta were stained with the Ab to the receptor and FITC-coupled secondary anti-rabbit Ab (ac) or with anti-renin Ab and Texas-red–coupled secondary Ab (eg). (d) FITC-secondary Ab alone. (h) TRITC–coupled secondary Ab alone. The receptor and renin have similar distribution in vascular structures and in syncytiotrophoblast cells.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
2.
Figure 7

Figure 7. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Expression of the receptor mRNA. Northern blot analysis. After hybridization of the human blot, a strong signal corresponding to a 2.4-kb mRNA band was detected in heart, brain, and placenta, a weaker signal was seen in liver, pancreas, and kidney. The mRNA of the receptor was hardly detected in lung and skeletal muscle. The amount of RNA on the membrane was adjusted so that β-actin hybridization is comparable in every lane.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
3.
Figure 2

Figure 2. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

In vitro transcription/translation of N14F/FLAG protein and coprecipitation with renin. (a) N14F/FLAG fusion protein (lane A) or control empty vector (lane B) transcribed in vitro and labeled with 35S-methionine are analyzed by SDS-PAGE and fluorography. (b) Coprecipitation of the N14F/FLAG fusion protein incubated with renin and immunoprecipitated with anti-renin Ab. The proteins were eluted and analyzed by SDS-PAGE and flurography. Lane A: N14F/FLAG fusion protein immunoprecipitated with anti-FLAG agarose; lane B: elution of N14F/FLAG protein bound to renin; lane C: N14F/FLAG protein immunoprecipitated with anti-renin Ab. (c) Renin alone (lane A) was immunoprecipitated with Ab to the renin receptor and analyzed by Western blotting with anti-renin Ab. Lane B: recombinant renin.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
4.
Figure 3

Figure 3. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Membrane expression of N14F protein and renin binding by HMC cells transformed with N14F. (a) Saturation of binding on the HMC clone 2 (HMC2) expressing the receptor (squares, total; circles, nonspecific) and on HMC control cells (+, total; ×, nonspecific). The nonspecific binding was determined in the presence of 100 nM cold renin. Inset: Scatchard plot of the binding of renin to HMC2. (b) Immunofluorescence and confocal analysis of the expression of N14F on HMC2 cells stably expressing N14F (upper panel) and by HMC control cells transfected with empty vector (lower panel). The cells were stained with anti-receptor Ab (dilution 1:1,000) and with TRITC–conjugated secondary Ab.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
5.
Figure 5

Figure 5. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Cross-linking with renin and phosphorylation of the receptor. (a) HMC and control cells were labeled with 35S-methionine, and the receptor was immunoprecipitated with anti-receptor Ab (lane A and lane B for HMC and control cells, respectively) or after incubation with renin and cross-linking (lane C and lane D for HMC and control cells, respectively). In lane E, HMC cells cross-linked in the absence of renin. (b) In the upper panel HMC2 cells were stimulated with renin in the presence of 100 nM Captopril. The receptor was immunoprecipitated with anti-FLAG agarose, and the eluate was analyzed by Western blotting using Ab’s to phosphotyrosine or to phosphoserine. In the lower panel control cells were stimulated with renin, and the cell lysate was analyzed by Western blotting using Ab’s to phosphotyrosine or to phosphoserine. The right lanes of the two lower blots are the receptor immunoprecipitated from HMC cells stimulated by renin.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
6.
Figure 4

Figure 4. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Ang I generation by soluble-phase and membrane-bound renin and prorenin. (a) Ang I generated by membrane-bound renin (squares, 1 nM; triangles, 0.5 nM) and by renin in solution phase (circles, 1 nM; diamonds, 0.5 nM). (b) Lineweaver-Burk plots of 1/Ang I generated vs. 1/angiotensinogen (Agen) by 1 nM renin bound to the membranes (squares) or in solution (circles), and by 0.5 nM renin on membranes (triangles) or in solution (diamonds). (c) Kinetic parameters of Ang I production by renin. (d) Comparison of Ang I generated by 0.5 nM renin and prorenin incubated with angiotensinogen (Agen) 1 μM under different conditions. Bars 1, 2, and 3 are renin in solution, pH 5.7, for 1 hour; pH 7.4 for 1 hour; and pH 7.4 for 4 hours, respectively. Bars 4 and 5 are prorenin pH 7.4 for 4 hours, in solution or membrane bound, respectively. Experiments were performed twice in duplicate, and the results represent the mean of the two experiments.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
7.
Figure 8

Figure 8. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Expression of the renin receptor on human kidney and heart. (a) At the left, immuofluorescence staining of kidney cortex with anti-receptor Ab (upper panel magnification ×20) and a glomerulus at higher magnification (lower panel, ×40). Yellow staining is due to autofluorescence of degradation products in tubular cells. The immune serum was preincubated with the peptides (right). (bm) Double-staining and analysis by immunofluorescence and confocal microscopy of kidney cortex labeled with anti-renin receptor (b, e, h), anti-CD31 (c, f, i), and anti-receptor plus anti-CD 31 (d, g, j). eg represent the glomerulus in bd, and hj represent the artery in bd at higher magnification. (km) A coronary artery labeled with anti-receptor, anti-CD31, anti-receptor plus anti-CD 31 Ab, respectively. (nv) Double-staining and analysis by immunofluorescence and confocal microscopy of kidney cortex labeled with anti-renin receptor (n and q) or smooth muscle α-actin Ab (SM-α-actin; o and r) and anti-receptor and anti–SM-α-actin (p and s); qs represent the kidney cortex artery in panels n and q at higher magnification. (tv) A coronary artery stained with anti-receptor, anti–SM-α-actin, and anti-receptor plus anti–SM-α-actin Ab, respectively.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
8.
Figure 1

Figure 1. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Nucleotide and amino acid sequence of N14F. (a) The nucleotide sequence is numbered on the right. The ATG of GCACCATGG is assigned as codon 1 on the basis of its close match to the C/GCACCATGG Kozak consensus sequence for optimal initiation of translation in eukaryotic cells. An in-frame TGA stop codon is located 858 nucleotides before the AATAAA cleavage and polyadenylation sequence, followed by the poly(A)+ sequence 15 nucleotides after the AATAAA. (b) The amino acid numbering begins with the first methionine of the longest open reading frame. The hydropathy profile of the deduced amino acid sequence of the N14F computed according to Kyte and Doolittle hydropathicity analysis is shown in the lower panel. The region with hydropathic index consistent with formation of a transmembrane spanning segment of 21 amino acids is boxed. Tyr 335, the amino acid most likely to be phosphorylated, is underlined.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.
9.
Figure 6

Figure 6. From: Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Intracellular calcium and cAMP changes, MAP kinases ERK1(p44)/ERK2(p42) activation induced by renin. (a) Cells expressing the receptor were stimulated by 100 nM renin, and the intracellular calcium changes were analyzed by spectrofluorometry (top). Cell stimulation by human thrombin (10 nM) was used as control. Analysis of intracellular cAMP changes by cells stimulated by 10 nM renin and by 1 μM PGE-1 or by 1 μM isoproterenol (Isop), as positive controls (bottom). The results represent the mean ± SD of two experiments performed in triplicate. *P < 0.05 compared with basal value. (b) HMC2 cells (left panel) or control cells (right panel) were stimulated with renin in the presence of 1 μM Losartan. At intervals, the cells were lysed and the lysate analyzed by Western blotting using Ab’s to active or to total ERK1 and ERK2. The blots were scanned and the ratio of active, phosphorylated ERK1/ERK2 to total ERK1/ERK2 was plotted using the NIH IMAGE program. MAP kinase activity assay was performed on HMC2 renin-stimulated cells (left bottom). The results are expressed as 32P incorporated and represent the mean ± SD of two experiments performed in triplicate. *P < 0.05 compared with basal value.

Genevieve Nguyen, et al. J Clin Invest. 2002 June 1;109(11):1417-1427.

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