Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Antibodies and markers used for immunohistochemistry.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Immunohistochemistry, Plasmid Preparation

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Top Panel (A–C): A low-magnification transverse section (5 µm) of the mouse kidney is shown. Discrete labeling is shown for staining for aquaporin-2 ( A. AQP2, red), a marker of the collecting ducts, SK3 ( B. SK3, green), and a merger of both channels ( C. Merge, yellow-organge for co-localization of AQP2 and SK3). Labeling is apparent for SK3 in both the cortex (label C) and medullary (label M) (dashed line shows cortical-medullary demarcation). Middle Pannel (D–F): Magnified view of the yellow inset box from A. SK3 co-localizes with all AQP2-postive tubules as show by the yellow-orange images (F., asterisk). SK3 staining is also apparent in AQP2-negative structures including other tubular structures (F., arrows) and smaller secondary structures (possibly vascular structures, F., arrow heads). Bottom Panel (G–H): Magnified view of staining in the presence of SK3 blocking peptide. All SK3 staining is abolished demonstrating specificity of our anti-SK3 antibody. Scale bar is 50 µm.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Labeling, Staining, Marker, Blocking Assay

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Section (5 µm) from WT mouse kidney showing staining for AQP2 (red), a marker of PCs in collecting duct, and SK3 (green). Panels A, C, and E are low magnification views of a cross-section through a CCD identified by AQP2 staining. Panels B, D, and F represent a magnified view of the inset area from A (yellow inset box). Panel B shows strong AQP2 staining along the luminal border of PCs (5–6 cells), but not of the ICs (2 cells without staining). As shown in D and F , strong staining of SK3 is evident along the luminal border of all cells, both PCs and ICs. Variable, but weak staining, is also apparent along the abluminal border of some cells. However, the staining is most pronounced along the luminal border for both PCs and ICs, although typically stronger in PCs, as indicated by the SK3 fluorescence line intensity profiles across (luminal to abluminal direction) two cells identified as PC and IC ( Panel G ). H . Relative mean intensity profiles (± SEM) across the cells from all sections showing the maximal values across the luminal border (Apical) and abluminal border (Basal) and the minimal values within the cytoplasm (Cytosol). The mean values are given for both PCs (n = 37) and ICs (n = 12) from all sections analyzed. The maximal luminal intensity is much greater than the abluminal intensity (*P<0.02) indicating dominant expression at the luminal border. Scale bar is 10 µm.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Staining, Marker, Fluorescence, Expressing

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Co-immunoprecipitation of aquaporin-2 (AQP2) and sorting nexin 27 (SNX27). ( A , B ) Immunoblotting of AQP2 in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune immunoglobulin G (IgG) of mouse (mIgG), Dynabead M-280 with anti-SNX27 antibody, pre-immune IgG of rabbit (rIgG), or Dynabead M-280 with anti-AQP2 antibody, respectively. ( C , D ) Immunoblotting of SNX27 in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune IgG of mouse (mIgG), Dynabead M-280 with anti-SNX27 antibody, pre-immune IgG of rabbit (rIgG), or Dynabead M-280 with anti-AQP2 antibody, respectively. ( E ) Immunoblotting of vacuolar protein sorting-associated protein 35 (Vps35) in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune IgG of rabbit (rIgG) or Dynabead M-280 with anti-SNX27 antibody, respectively. ( F ) Human Embryonic Kidney 293T (HEK293T) cells were transiently expressed with hemagglutinin (HA)-tagged AQP2 (full length) plasmid alone or both HA-tagged AQP2 (full length) and FLAG-tagged SNX27 (full length) plasmid. Immunoblotting of AQP2 and SNX27. In the control condition (Con), cells were transfected only with p3XFLAG-CMV-10 and pcDNA3.1-HA. ( G ) Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-AQP2 antibody and anti-SNX27 antibody. ( H ) Schematic representation of SNX27 constructs. ( I ) Immunoblotting using anti-glutathione S-transferase (GST) antibody after purification of GST-tagged SNX27 constructs. ( J ) Immunoblotting using anti-AQP2 antibody after purification of histidine (His)-tagged carboxyl terminus of AQP2 (AQP2c). ( K , L ) GST-SNX27 fusion proteins were incubated with His-tagged AQP2c proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted with anti-glutathione S-transferase (GST) or anti-AQP2 antibody.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Immunoprecipitation, Western Blot, Suspension, Plasmid Preparation, Control, Transfection, Construct, Purification, Incubation

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Immunofluorescence of SNX27 and AQP2 in HeLa cells. FLAG-tagged SNX27 and HA-tagged AQP2 constructs were transiently transfected into HeLa cells and immunolabeling of FLAG and HA was done. A negative control study revealed no immunolabeling of FLAG and HA in the HeLa cells transiently transfected with SNX27-full length and HA-tagged AQP2 constructs, which were incubated only with secondary antibody (omitting the incubation of primary antibodies) ( A – C ). SNX27 (SNX27-Full Length, Δ(PX+FERM), and ΔFERM) and AQP2 were co-localized throughout the cytoplasm in HeLa cells ( D – L ). In contrast, when the PDZ domain was deleted in SNX27 (SNX27-ΔPDZ), SNX27 and AQP2 accumulated in a punctate pattern, and more eccentrically localized in the perinuclear region of the HeLa cells ( M – O ). ( P ) Co-localization of SNX and AQP2 was analyzed by calculation of the Pearson’s coefficient. Graphs express means ± SE (>20 cells per group; three independent experiments). Scale bars, 10 μm.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Immunofluorescence, Construct, Transfection, Immunolabeling, Negative Control, Incubation

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Immunofluorescence of AQP2, Lysotracker, or GM130 in HeLa cells. FLAG-tagged SNX27 and HA-tagged AQP2 constructs were transiently transfected into HeLa cells. ( A – H ) Cells were stained with Lysotracker (red), followed by immunolabeling of HA (green). Diffuse cytoplasmic HA labeling (i.e., AQP2 in panels ( A – F )) was not or weakly overlaid by Lysotracker Red staining in the cytoplasm of HeLa cells with PDZ domain-expressing SNX27 ( A – F ). In contrast, when the PDZ domain was deleted in SNX27 (SNX27-ΔPDZ), the punctate AQP2 labeling was observed, which was intensively overlaid by Lysotracker Red staining ( G , H ). Panels ( B , D , F , H ) are zoomed-in images of panels ( A , C , E , G ), respectively. ( I – T ) Cells were co-immunolabeled with anti-HA and anti-GM130 antibodies. HA-AQP2 labeling was not overlaid by GM130 labeling in the presence or absence of the PDZ domain in SNX27. ( U , V ) Co-localization of HA-tagged AQP2 and Lysotracker ( U ) or HA-tagged AQP2 and GM130 ( V ) was analyzed by calculation of the Pearson’s coefficient. Graphs express means ± SE (>30 cells per group; three independent experiments). * p < 0.05. Scale bars, 10 μm.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Immunofluorescence, Construct, Transfection, Staining, Immunolabeling, Labeling, Expressing

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Immunofluorescence microscopy of AQP2 and SNX27 in the rat kidney inner medulla. Immunofluorescence labeling of AQP2 in the inner medullary collecting duct cells of the kidney from vehicle-treated control rats (Control, ( A )), rats with dDAVP infusion for 5 days (dDAVP, ( D )), and rats with dDAVP withdrawal for 3 h after dDAVP infusion for 5 days (dDAVP/withdrawal, ( G )). SNX27 immunolabeling in the kidneys from vehicle-treated rats ( B ), rats with dDAVP infusion for 5 days (D5d, ( E )), and rats with dDAVP withdrawal for 3 h after dDAVP infusion for 5 days (D5d-3 h, ( H )). Immunofluorescence labeling of AQP2 and SXN27 was merged ( C , F , I ). ( J ) Co-localization of AQP2 and SNX27 was analyzed by calculation of the Pearson’s coefficient. Graphs express means ± SE (>200 cells in the collecting ducts per group; two independent experiments). Scale bars, 10 μm.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Immunofluorescence, Microscopy, Labeling, Control, Immunolabeling

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Immunofluorescence microscopy of AQP2 and SNX27 in primary cultured inner medullary collecting duct (IMCD) cells of the rat kidney. The immunofluorescence labeling of AQP2 and SNX27 in vehicle-treated IMCD cells (( A , B ) Control), dDAVP (10 −9 M)-treated IMCD cells for 24 h (( D , E ) dDAVP), and IMCD cells with dDAVP withdrawal for 3 h after dDAVP (10 −9 M)-treatment for 24 h (( G , H ) dDAVP-3 h). Immunofluorescence labeling of AQP2 and SXN27 was merged ( C , F , I ). ( J ) Co-localization of AQP2 and SNX27 was analyzed by calculation of the Pearson’s coefficient. Graphs express means ± SE (>200 cells per group; two independent experiments). * p < 0.05. Scale bars, 10 μm.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Immunofluorescence, Microscopy, Cell Culture, Labeling, Control

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Semiquantitative immunoblotting and quantitative real-time PCR of SNX27 and AQP2 in mpkCCDc14 cells with siRNA-mediated SNX27 knockdown. ( A – C ) Semiquantitative immunoblotting of SNX27 (~65 kDa) and AQP2 (~29 kDa and ~35–50 kDa) in total cell lysates from mpkCCDc14 cells treated with vehicle or dDAVP (10 −9 M) for 24 h under control siRNA or SNX27-siRNA transfection. n indicates the number of cell preparations from three independent experiments. ( D , E ) SNX27 and AQP2 mRNA level in mpkCCDc14 cells treated with vehicle or dDAVP (10 −9 M) for 24 h under control siRNA or SNX27-siRNA transfection. n indicates the number of cell preparations from two independent experiments. * p < 0.05.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Control, Transfection

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Semiquantitative immunoblotting of SNX27 ( A , B ; F , G ; and K , L ) and AQP2 ( A , C ; F , H ; and K , M ) in total cell lysates from mpkCCDc14 cells transfected with control-siRNA or SNX27-siRNA. Semiquantitative immunoblotting of AQP2 in total cell lysates from mpkCCDc14 cells transfected with control siRNA or SNX27-siRNA subjected to 24-h dDAVP stimulation (10 −9 M) or 3-h withdrawal (Withdrawal) after dDAVP stimulation (10 −9 M, 24 h) in the absence (-) or the presence (+) of chloroquine (10 −4 M, for the last 3 h, ( A , D , E )), bafilomycin (10 −7 M, for the last 3 h, ( F , I , J )), or MG-132 treatment (MG-132, 10 −6 M, for the last 3 h, ( K , N , O )). n indicates the number of cell preparations from three independent experiments. D, dDAVP. * p < 0.05.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Western Blot, Transfection, Control

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: Cell surface biotinylation assay of AQP2 in mpkCCDc14 cells with siRNA-mediated SNX27 knockdown under dDAVP stimulation. ( A – C ) Semiquantitative immunoblotting of SNX27 (~65 kDa) and AQP2 (~29 kDa and ~35–50 kDa) in the total cell lysates or biotinylated fraction from mpkCCDc14 cells transfected with control siRNA or SNX27-siRNA. ( A , D ) Cell surface biotinylation assay for examining the changes in dDAVP (10 −9 M, 24 h)-induced AQP2 expression in the apical plasma membrane of the mpkCCDc14 cells. ( E ) Immunofluorescence microscopy of AQP2 in mpkCCDc14 transfected with control siRNA or SNX27-siRNA, followed by dDAVP treatment (10 −9 M, 24 h). In the x-z images, arrows indicate AQP2 expression in the apical plasma membrane. n indicates the number of cell preparations from three independent experiments. * p < 0.05 when compared to control siRNA-transfected cells.

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Cell Surface Biotinylation Assay, Knockdown, Western Blot, Transfection, Control, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Microscopy

Journal: Cells

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

doi: 10.3390/cells9051208

Figure Lengend Snippet: A summary of the findings in the present study. Upon the withdrawal of vasopressin stimulation, AQP2 is internalized into early endosomes for sorting. At the early endosome, AQP2 could be entered (1) to the recycling pathways either via the trans-Golgi network or directly to the plasma membrane, leading to AQP2 expression in the apical plasma membrane of the collecting duct principal cells in the kidney or (2) to the lysosomal pathway for degradation, leading to downregulation of AQP2 in the plasma membrane and the cells [ , , , , , , , , ]. The class I PDZ domain-binding motif (X-[S/T]-X-Φ) in the carboxyl terminus of AQP2 is recognized by PDZ domain-containing proteins, e.g., SNX27. SNX27 is bound to the Vps26 of retromer complex subunits and concurrently binds to the PDZ ligand in its cargo proteins (AQP2); thereby, it could be involved in the recycling of AQP2 into the plasma membrane. The retromer complex is involved in the retrograde transport of proteins from endosomes to the trans-Golgi network. Alternatively, AQP2 could be sorted into lysosomes and subjected to lysosomal degradation to a greater extent upon the suppression of SNX27, i.e., SNX27 knockdown or deletion of the PDZ domain and Vps35 depletion .

Article Snippet: For double-immunolabeling of AQP2 and SNX27 on kidney sections, deparaffinized sections were incubated with rabbit anti-AQP2 polyclonal antibody (1:200, AB3274, Merck Millipore), and mouse anti-SNX27 monoclonal antibody (1:200, ab77799, abcam) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG.

Techniques: Clinical Proteomics, Membrane, Expressing, Binding Assay, Knockdown

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Apelin-13 Regulates Vasopressin-Induced Aquaporin-2 Expression and Trafficking in Kidney Collecting Duct Cells.

doi: 10.33594/000000165

Figure Lengend Snippet: Fig. 6. Representative immunofluorescence images showing the effect of apelin-13 on AQP2-pS269 localization in mpkCCDc14 cells. Cells grown on permeable supports were left untreated (negative control, 1st column) or treated with either dDAVP for 24h (positive control, 2nd column) or with a combination of apelin (for 30 or 60 min, 3rd and 4th columns) and dDAVP. Double immunofluorescence labeling of AQP2-pS269 (red) and occludin (green) was performed using an anti-AQP2-pS269 and anti-OCLN. Nuclei were stained with DAPI (blue). Confocal images are shown in both XY and XZ plans. Scale bar = 5 µm. Localization analysis of AQP2-pS269 represents the means ± SEM of four independent experiments compared to dDAP treatment. §§§ p< 0.001; ***p<0.001 two-way ANOVA with a post-hoc Bonferroni multiple comparison test.

Article Snippet: Filters were then incubated with either rabbit polyclonal anti-AQP2 primary antibody (1/500, Novus) or rabbit anti-AQP2-pS269 antibody (1/600, Phosphosolution).

Techniques: Immunofluorescence, Negative Control, Positive Control, Labeling, Staining, Comparison

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 1. The increased level of AQP2 with thirsting was significantly attenuated in CD-KO mice. (A) AT1A receptor mRNA expression is reduced in the renal inner medulla of CD-KO mice compared with control mice as determined by real time PCR (1.00 0.09 versus 0.56 0.07; n 12; *P 0.01). No differences in AT1A receptor expression were detected in the cortex and the outer medulla of CD-KO and control mice. Representative confocal laser-scanning microscopy (20 and 40) of the inner medulla of Hoxb7-Cre

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Confocal Laser Scanning Microscopy

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 3. Increases of AQP2 with thirsting are significantly attenuated in CD-KO mice compared with controls. Representative immunoblots and densitometric analysis of aquaporin-2 (AQP2) in renal outer and inner medulla in CD-KO and control mice at baseline (control: n 4; CD-KO: n 4) and after 18 hours of water deprivation (control: n 4; CD-KO: n 5). In water-deprived control mice, outer medullary AQP2 expression levels were significantly increased compared with baseline controls and water-deprived CD-KO mice. (A) Outer medullary AQP2 abundance tended to be increased in water-deprived CD-KO mice compared with baseline. (B) Inner medullary AQP2 expression levels were similar between CD-KO and control mice at baseline. After 18 hours of water deprivation, inner medulla AQP2 expression levels increased significantly in both groups. AQP2 expression levels were significantly lower in the inner medulla of CD-KO mice compared with control mice. For the densitometric analysis, AQP2 expression levels were normalized to the corresponding -actin expression and further evaluated by calculating the fold induction relative to the AQP2/-actin ratio observed in the sample from mouse #1 (baseline control). The sample from mouse #1 is present as a reference on all Western blots to allow comparison of all samples. †P 0.01 versus baseline control; #P 0.05 versus baseline CD-KO; *P 0.05 versus water deprivation control. The data are presented as the means SEM.

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Western Blot, Control, Expressing, Comparison

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 4. Normal cellular localization of AQP2 in CD-KO mice. Representative confocal laser-scanning microscopy (64) of the inner medulla from CD-KO and control mice at baseline and after 18 hours of water deprivation. Under baseline conditions, aquaporin-2 (AQP2) (red) is diffusely localized around the apical membrane of the collecting duct in control (A and E) and CD-KO mice (B and F). After 18 hours of water deprivation, intense AQP2 expression strictly defined to the apical membrane of principle cells were detected in both control (C and G) and CD-KO (D and H) mice (C and D). However, similar to AQP2 protein expression levels (Figure 3), the extent of apical AQP2 labeling after 18 hours of water deprivation appeared to be stronger in control mice compared with CD-KO mice. DAPI, 4,6,diamidino-2-phenylindole; WD, water deprivation.

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Confocal Laser Scanning Microscopy, Control, Membrane, Expressing, Labeling

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 5. AT1A receptor mRNA expression is reduced in the renal inner medulla of CD-KO (AQP2-Cre) mice compared with control mice as determined by real time PCR (1 0.08 versus 0.49 0.09; *P 0.05; n 4 to 8). (A) No differences in AT1A receptor expression were detected in the cortex of CD-KO (AQP2-Cre) and control mice (cortex 1.00 0.1285 versus 0.89 0.11; P 0.56; n 4 to 8). Basal urine osmolalities in CD-KO (AQP2-Cre) and control mice were similar (1120 41 versus 1114 18 mOsmol/ kg; n 8). After 18 hours of water deprivation, urine osmolality increased significantly in CD-KO (AQP2-Cre) and control mice (*P 0.001). (B) Urine osmolalities remained significantly lower in CD-KO (AQP2-Cre) mice compared with control mice (3081 90 versus 3535 147 mOsmol/kg; #P 0.05; n 9). The data are presented as the means SEM.

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction