1. Ephrin-B1 localizes at the slit diaphragm of the glomerular podocyte
T. Hashimoto, T. Karasawa, A. Saito, N. Miyauchi, G.D. Han, K. Hayasaka, F. Shimizu, H. Kawachi 
Kidney International 
Volume 72, Issue 8, Pages (October 2007)
DOI: /sj.ki
Copyright © 2007 International Society of Nephrology Terms and Conditions

2. Figure 1
Reverse-transcription-(RT)-PCR findings of EphBs and ephrin-Bs, and cloning of rat homologues of ephrin-B2 and EphB2. (a) RT-PCR findings of EphBs and ephrin-Bs. The sizes of the PCR products for EphB1, EphB2, ephrin-B1 and ephrin-B2 detected in glomeruli (G) and in cultured podocytes (P) are the same as those in brain (B) samples. The PCR products corresponding to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) are shown at the bottom. (b) Comparison of the amino-acid sequences of rat, mouse, and human ephrin-B2. The dark gray boxes indicate the conserved amino acids shared by rat, mouse, and human ephrin-B2. The gray boxes indicate the conserved amino acids shared by ephrin-B2 from two of the three species. The identity of the amino-acid sequence of rat and mouse ephrin-B2 is 98.8%. The identity of the amino-acid sequence of rat and human ephrin-B2 is 97.3%. The sequence data of rat ephrin-B2 we cloned are available from GenBank under accession number DQ (c) Comparison of the amino-acid sequences of rat and human EphB2. The gray boxes indicate the conserved amino acids shared by rat and human EphB2. The identity of the amino-acid sequence of rat and human EphB2 is 98.9%. The sequence data of rat EphB2 that we cloned are available from GenBank under accession number DQ (d) Semi-quantitative RT-PCR findings of EphB1, EphB2, ephrin-B1, and ephrin-B2 in ANA-induced nephropathy (ANA). The ratios of the densitometric signal to that of GAPDH were analyzed; the data are shown as ratios relative to the normal findings and are expressed as means±s.d. of three independent experiments. Representative agalose gel electrophoretic patterns from one of three experiments are shown at the bottom. The mRNA expression of EphB2 and ephrin-B1 decreased at 24 h of ANA nephropathy (EphB2: 58.5±9.0%, ephrin-B1: 48.9±11.5%).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

3. Figure 2
Expression of ephrin-B1 in the glomeruli. (a) Western blot findings of ephrin-B1 in rat glomerular lysate. Positive bands of around 50 kDa were detected with anti-ephrin-B1 antibody (rabbit) (lane 1). Bands were not detected with normal rabbit serum (lane 2). The ephrin-B1-specific bands were not detected in the stripe stained with the antibody pre-incubated with its immunizing peptide (lane 3). Positive bands were detected with the antibody preincubated with the same amount of irrelevant peptide (lane 4). (b–h) IF findings of ephrin-B1 in rat glomeruli. (b) The staining of anti-ephrin-B1 antibody (rabbit) was detected along the glomerular capillary loop. (c) Negative staining was detected with normal rabbit serum. (d) No positive staining was detected with anti-ephrin-B1 antibody preabsorbed with the peptide used for immunization. The intensity of the staining with the antibody preincubated with the ephrin-B1-specific peptide no. 1 (e) was clearly lower than that with the antibody without pre-incubation. The preincubation with the ephrin-B1-specific peptide no. 2 (f), no. 3 (g), or an irrelevant peptide (h) did not reduced the intensity of the staining (original magnification × 400). (i–j) IF findings of ephrin-B1 in human glomeruli. (i) The staining of anti-ephrin-B1 antibody (rabbit) along the glomerular capillary loop was also observed in human kidney section. (j) No positive staining was detected with normal rabbit serum (original magnification × 400).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

4. Figure 3
Localization of ephrin-B1 in rat glomeruli. (a–l) Dual-labeling IF findings of ephrin-B1 (a–d, green) with glomerular cell markers (i–l, red) in adult rat sections (e–h, merged image). Anti-rat endothelial cell antigen (RECA-1) antibody and anti-Thy1.1 antibody (mAb ) were used as an endothelial cell marker (i) and a mesangial cell marker (j), respectively. ANA (mAb 5-1-6) (k) and anti-CD2-associated protein (CD2AP) antibody (goat) (l) were used as podocyte markers. The staining of anti-ephrin-B1 antibody (rabbit) was clearly apart from the RECA-1 (e) and Thy1.1 (f) staining. The ephrin-B1 staining colocalized with the nephrin staining (g), and with the CD2AP staining (h) (original magnification, × 400). (m–o) Immunoelectron microscopic findings. The gold particles for the anti-ephrin-B1 antibody (goat) were mainly detected at the slit diaphragm (arrows) (m–o), although some gold particles were detected at the basal part of podocytes (m) (original magnification: m, n: × 39 380; o: × 78 750). (p–s) Dual-labeling IF findings of anti-ephrin-B1antibody (green) with ANA (red) in embryonic (p, q: embryonic day 20.5) and infantile (r, s) rat kidney sections. Ephrin-B1staining was clearly apart from nephrin staining at S-shaped body stage (p) and at the early capillary loop stage (q). Some portions of ephrin-B1 staining colocalized with nephrin staining at the late capillary loop stage (r) and at the developing stage (s) (arrows) (original magnification, × 400). (t–y) Interaction of ephrin-B1 with nephrin. Immunoprecipitation of anti-nephrin-antibody (rabbit) was performed with normal glomerular lysate solubilized by radioimmunoprecipitation assay buffer (lanes 1, 3). Immunoprecipitation of preimmune normal rabbit serum (NRS) was carried out as a control (lanes 2, 4). Western blot analysis with the precipitated materials was performed with ANA (rabbit) (t), anti-CD2AP antibody (rabbit) (u), anti-ephrin-B1 antibody (goat) (v), preimmune normal rabbit serum (w, x) and normal goat serum (NGS) (y). The specific ephrin-B1 band (lane 1-v) and the CD2AP band (lane 1-u) were detected in the immunoprecipitate of the ANA. No band was detected in the precipitates of normal rabbit serum (lanes 2-t, 2-u, 2-v and lanes 4-w, 4-x, 4-y). No band was detected with normal rabbit or goat serum in the precipitates of the ANA (lanes 3-w, 3-x, 3-f).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

5. Figure 4
Ephrin-B1 expression in ANA-induced nephropathy. (a–q) IF findings of anti-ephrin-B1antibody in ANA nephropathy. The staining of ephrin-B1 in normal, 24 h of ANA nephropathy (ANA 24 h) and 5 days of ANA nephropathy glomeruli (ANA 5 day) was evaluated as described in Materials and Methods section. Representative glomeruli showing the score 0–4 were shown in the upper panel (a–e). The graphs showing % of the glomeruli of each score to total number of the glomeruli are shown in the left panel (f–h). The data are shown as mean±s.d. (n=5 rats). Representative IF findings of anti-ephrin-B1 antibody (low (i–k) and high (l–n) magnification) were shown in the middle panel (i–n). In normal rat glomeruli, the specific ephrin-B1 staining was detected along capillary loop in all tuft area, and more than 80% of glomeruli were classified as score 3 or 4 (i, l). The staining intensity of ephrin-B1 significantly decreased at 24 h (j, m), and it recovered to normal level on day 5 (k, n). The representative nephrin stainings of each group were shown in the right panel (o–q). The alteration of nephrin staining is not remarkable at 24 h (p), although the nephrin staining clearly shifted to a discontinuous coarse granular pattern on day 5 (q) (original magnification, i–k: × 100; l–q: × 400). (r) Western blot findings of anti-ephrin-B1 antibody (rabbit) in glomerular lysate solubilized with SDS-PAGE sample buffer for rats at 24 h of ANA nephropathy. The ratios of the densitometric signal of ephrin-B1 to that of the internal control actin were analyzed. The data are shown as ratios relative to the normal findings and are expressed as means±s.d. of three independent experiments. Representative Western blotting patterns from one of three experiments are shown. The amount of ephrin-B1 reduced at 24 h of ANA nephropathy (73.2±14.8% to normal).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

6. Figure 5
Expression of ephrin-B1 in cultured podocytes, and RNA silencing analysis. (a–d) Expression of ephrin-B1 in cultured podocytes. Anti-ephrin-B1 antibody (rabbit) staining was detected in the cultured podocytes (a). No positive staining was detected with normal rabbit serum (b) or with the anti-ephrin-B1 antibody pre-absorbed with the peptide used for immunization (c). Positive staining was detected with the antibody pre-incubated with the same amount of irrelevant peptide (d) (original magnification, × 200). (e–p) Analysis of gene silencing for ephrin-B1. (e) mRNA expression of ephrin-B1 in the cultured podocytes treated with ephrin-B1 small interfering RNA (siRNA) for 48 h. GAPDH was analyzed as the internal control. The data are shown as the ratio (%) relative to the control siRNA-treated cells, and are expressed as means±s.d. of three independent experiments. Representative agalose gel electrophoretic patterns from one of three experiments are shown. mRNA expression of ephrin-B1 clearly decreased in the material treated with siRNA for ephrin-B1 (29.7±17.3%). (f, g) Immunohistological analysis. The intensity of ephrin-B1 was clearly lower in the cells treated with ephrin-B1 siRNA (g) than that in the cells treated with the control siRNA (f). (h) Western blot analysis with cell lysate solubilized with SDS-PAGE sample buffer. The data are shown as a ratio (%) relative to the control siRNA-treated cells, and are expressed as means±s.d. of three independent experiments. The intensity of the band of ephrin-B1 was decreased in the material treated with siRNA for ephrin-B1 (66.9±7.6%). (i–p) Immunohistological analysis in cultured podocytes treated with control siRNA (i–l) or ephrin-B1 siRNA (m–p). Dual-labeling IF findings of anti-ephrin-B1 antibody (i, m) with anti-CD2AP antibody (k, o) and merged imaged (j, n), and the single-labeling IF findings of F-actin staining (l, p) are shown. EphrinB-1 signals radiating from the perinuclear area towards the tip end of the processes were detected in the cells treated with control siRNA (i, arrows). CD2AP signals were also detected at the processes in the cells treated with control siRNA (k, arrows). The intensity of ephrin-B1 staining was reduced in the cells treated with ephrin-B1 siRNA (m). CD2AP signals at the processes were not detected in the cells treated with ephrin-B1 siRNA. CD2AP signals were limitedly detected around the nuclei (o, arrows). No significant difference in F-actin staining was detected between the cells treated with control siRNA and those treated with ephrin-B1 siRNA (l, p) (original magnification, × 200).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions