Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Gpr116 is highly expressed in kidney and is localized to V-ATPase expressing ICs of the collecting duct. (A) Gpr116 mRNA expression as measured by qRT-PCR in various tissues including lung and kidney. SP-C and AQP2 served as positive controls of lung and kidney mRNA. Gene expression is normalized to Gapdh. Cycle threshold of 35 approximately corresponds with 0.001 on the y axis. (B) Western blot analysis of Gpr116 expression in various tissues as well as stably transfected HEK293 cells with mouse (mGpr116) or human (hGpr116) Gpr116. Expected molecular mass for Gpr116 is ∼150 kDa. (C) Diagram of the murine outer medullary collecting duct (OMCD) showing water-transporting principal cells (PC) and acid-secreting A-IC. Proton secretion is coupled to bicarbonate reabsorption throughout the nephron. (D) Representative immunofluorescence images demonstrating colocalization of Gpr116 with V-ATPase in mouse collecting ducts. Similarly, Gpr116 is interspersed among AQP2 expressing cells in mouse collecting ducts. (Scale bars, 20 μm.)

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Stable Transfection, Transfection, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Targeted deletion of Gpr116 in kidney tubules results in loss of Gpr116 in collecting ducts. (A) Representative immunofluorescence images demonstrating localization of Gpr116-expressing cells among AQP2 expressing cells in WT mouse collecting ducts. (B) Representative immunofluorescence images demonstrating loss of Gpr116-expressing cells in mouse collecting ducts after targeted deletion with KSP-Cre/Lox system. (Scale bars, 20 μm.)

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques: Immunofluorescence, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: KO mice do not have more A-ICs than WT mice. (A) Representative images demonstrating AQP2 (green) labeling of principal cells and AE1 (red) labeling of ICs from mice drinking control water and water with NH4Cl. (Scale bars, 20 μm.) (B, Left) Quantification of AE1+ cells per 100 DAPI+ cells. For each kidney section, quantification was performed on whole medulla. (Right) Quantification of AQP2+ cells per 100 DAPI+ cells. For B, bars are mean ± SEM. n = 6 kidneys per group. Solid bars are 0.5% sucrose water, hatched bars are 0.5% sucrose + 280 mM NH4Cl water.

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques: Labeling, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: mRNAs of V-ATPase subunits are similar in WT and KO mice. qPCR analysis of AQP2, SLC4A1 (AE1), V-ATPase subunits ATP6V1B1, ATP6V0D2, ATP6V1G3, AQP6, and CLCN5 mRNAs are not statistically different in WT and KO kidney samples. V-ATPase subunits ATP6V0D2 and ATP6V1G3 were previously shown to be unique transcriptional markers for A-ICs in the kidney (18). AQP6 and CLCN5 encode for proteins that colocalize with V-ATPase in subapical vesicles (59). Bars are mean ± SEM. n = 4 WT kidneys, 3 KO kidneys. Cycle threshold of 35 approximately corresponds with 0.001 on the y axis.

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Gpr116 activation in A-ICs inhibits pHi recovery. (A) Representative pseudocolor images (blue, acidic; red, alkali) of intracellular pH in a split opened collecting duct loaded with pH-sensitive dye BCECF at the baseline (1), upon application of 40 mM NH4Cl (2), immediately after NH4Cl removal (3), and recovery toward the baseline pHi values (4). Confocal micrograph of the same split-opened CD probed with anti-AQP2 (pseudocolor red) is shown on the right. Examples of AQP2− ICs are depicted with white arrows. Nuclear DAPI staining is shown in pseudocolor blue. Magnification: 40×. (B) Summary graph comparing the time course of pHi changes in control (black) and p16 pretreated (100 µM for 40 min, red) ICs. Trace shows mean ± SEM for 65 control cells and 69 p16-treated cells. Each experimental condition is a summary from four collecting ducts from four different mice. Application of the NH4Cl pulse is designated by the black bar above the trace. The time points shown in A are marked as 1 to 4. (C) Summary graph of H+ extrusion rate from ICs from the control and after pretreatment with p16 shown in B. The rate was calculated as a linear slope of pHi recovery after 40 mM NH4Cl application. Statistical analysis performed using Mann–Whitney test. *P < 0.05. Control vs. p16 *P < 0.0001.

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques: Activation Assay, Staining, Control, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques: Diagnostic Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: TaqMan real-time PCR assays used

Article Snippet: Antibodies used in the study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Gene TaqMan ID Exon boundary ADGRF5 (Gpr116) Mm00685646_m1 6–7 SFTPC (SP-C) Mm00488144_m1 1–2 AQP2 Mm00437575_m1 1–2 SLC4A1 (AE1) Mm00441492_m1 3–4 ATP6V1B1 Mm00460309_m1 2–3 ATP6V0D2 Mm01222963_m1 1–2 ATP6V1G3 Mm00616840_m1 2–3 AQP6 Mm00558232_m1 1–2 CLCN5 (ClC-5) Mm00443851_m1 8–9 Open in a separate window TaqMan real-time PCR assays used Animals.

Techniques: Real-time Polymerase Chain Reaction

Journal: Histochemistry and cell biology

Article Title: Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

doi: 10.1007/s00418-010-0741-7

Figure Lengend Snippet: Tubular location of TRA-1-60 epitope in the cortex of human kidney. Sections of human renal cortex were stained in series with antibodies against EMA (a, e, i), THP (b, f, j), TRA-1-60 (c, g, k) and aquaporin-2 (d, h, l). TRA-1-60 staining (c, g, k arrows) corresponded positively with EMA (a, e, i arrows) and THP staining (b, f, j arrows) and negatively with aquaporin-2 (d, h, l arrows). Some tubules showed THP staining cytoplasmically and apically indicative of TAL (f and j arrows). Some tubules showed THP staining only at the apical surface, most likely representing distal convoluted tubules (b arrows). Scale bar 30 μm

Article Snippet: Primary mouse monoclonal anti-TRA-1-60 (Millipore, UK); anti-human epithelial membrane antigen (EMA, Dako Cytomation), clone E29, anti-human Ki-67 antigen (Dako Cytomation), clone MIB-1, rabbit anti-aquaporin 2 (Santa Cruz Biotech), rabbit anti-aquaporin-2 (Lifespan Biosciences) and sheep anti-Tamm–Horsfall glycoprotein (THP) (Millipore, UK) were used in single or dual immunostaining experiments of foetal and adult tissues with rabbit polyclonal anti-murine Pax2 antibody (Invitrogen) and anti-human Ki-67 antigen (Abcam).

Techniques: Staining

Journal: Histochemistry and cell biology

Article Title: Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

doi: 10.1007/s00418-010-0741-7

Figure Lengend Snippet: TRA-1-60 epitope localisation in specific areas of human medulla and papilla. Sections of human renal inner medulla/papilla and outer medulla were stained in series with antibodies against EMA (a, e, i, m, q), THP (b, f, j, n, r), TRA-1-60 (c, g, k, o, s) and aquaporin-2 (d, h, l, p, t). In the inner medulla/papilla (a–d) TRA-1-60 staining (c) corresponded to small thin-walled, EMA-positive tubules in the thin limb of the loop of Henle (arrowheads) and also to collecting ducts that were positive for aquaporin-2 (d) and EMA (a arrows). Here both types of tubules were negative for THP (b). In the inner stripe of the outer medulla (e–h; i–l and m–p), TRA-1-60 was localised to the collecting ducts (G arrows) corresponding positively with staining for EMA (e arrows) and aquaporin-2 (h arrows) and negatively with THP (f arrows), to the TAL (k arrow), corresponding positively with EMA (i arrow) and THP (j arrow) and negatively with aquaporin-2 staining (l arrow) and to the thin limb of the loop of Henle (o arrow) stained positively for EMA (m arrow) and negatively for THP (n arrow) and aquaporin-2 (p arrow). In the outer stripe of the outer medulla (q–t), TRA-1-60 was expressed in TAL (s arrow) corresponding positively with EMA (q arrow) and THP (r arrow) and negatively with aquaporin-2 (t arrow). Scale bar 60 μm (a and e), 30 μm (i, m and q)

Article Snippet: Primary mouse monoclonal anti-TRA-1-60 (Millipore, UK); anti-human epithelial membrane antigen (EMA, Dako Cytomation), clone E29, anti-human Ki-67 antigen (Dako Cytomation), clone MIB-1, rabbit anti-aquaporin 2 (Santa Cruz Biotech), rabbit anti-aquaporin-2 (Lifespan Biosciences) and sheep anti-Tamm–Horsfall glycoprotein (THP) (Millipore, UK) were used in single or dual immunostaining experiments of foetal and adult tissues with rabbit polyclonal anti-murine Pax2 antibody (Invitrogen) and anti-human Ki-67 antigen (Abcam).

Techniques: Staining

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: Clinical and Experimental Nephrology

Article Title: Phosphorylation profile of human AQP2 in urinary exosomes by LC–MS/MS phosphoproteomic analysis

doi: 10.1007/s10157-020-01899-4

Figure Lengend Snippet: The frequency of phosphorylated sites in all phosphorylated AQP2 PSM

Article Snippet: The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [ ], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [ , ], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [ ], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [ ] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [ ].

Techniques:

Journal: Clinical and Experimental Nephrology

Article Title: Phosphorylation profile of human AQP2 in urinary exosomes by LC–MS/MS phosphoproteomic analysis

doi: 10.1007/s10157-020-01899-4

Figure Lengend Snippet: Western blots of urinary exosomes with total-, pS256-, pS261-, pS264- and pS269-AQP2 specific antibodies. The same amounts of urinary exosome samples prepared from human ( a ) and mouse ( b ) were loaded in each lane

Article Snippet: The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [ ], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [ , ], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [ ], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [ ] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [ ].

Techniques: Western Blot

Journal: Clinical and Experimental Nephrology

Article Title: Phosphorylation profile of human AQP2 in urinary exosomes by LC–MS/MS phosphoproteomic analysis

doi: 10.1007/s10157-020-01899-4

Figure Lengend Snippet: Effects of dDAVP administration in a CDI patient. A. Urine osmolality and creatinine concentrations. B. Urinary AQP2 excretion was corrected by urinary creatinine (AQP2 concentration/creatine concentration)

Article Snippet: The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [ ], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [ , ], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [ ], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [ ] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [ ].

Techniques: Concentration Assay

Journal: Clinical and Experimental Nephrology

Article Title: Phosphorylation profile of human AQP2 in urinary exosomes by LC–MS/MS phosphoproteomic analysis

doi: 10.1007/s10157-020-01899-4

Figure Lengend Snippet: Western blots of urinary exosomes of CDI patient before and after dDAVP treatment. Left, blots were stained with total-, pS256-, pS261- and pS269-AQP2 specific antibodies. Exosome samples equivalent to 2 µg urine creatinine were applied in each lane with duplicate for each time point. Right, the band intensities of the blots were quantified and summarized. For each time point, the mean of two lanes was used

Article Snippet: The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [ ], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [ , ], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [ ], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [ ] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [ ].

Techniques: Western Blot, Staining

Journal: Clinical and Experimental Nephrology

Article Title: Phosphorylation profile of human AQP2 in urinary exosomes by LC–MS/MS phosphoproteomic analysis

doi: 10.1007/s10157-020-01899-4

Figure Lengend Snippet: Differential roles of phosphorylations at the C-terminal of AQP2 in its trafficking. Only the phosphorylations at S261 and T269 are marked with red filled circles

Article Snippet: The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [ ], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [ , ], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [ ], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [ ] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [ ].

Techniques:

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