polyclonal anti-aqp2 antibody  (Alpha Diagnostics)

Journal: Life

Article Title: Duloxetine-Induced Antidiuresis in Rats with Lithium-Induced Nephrogenic Diabetes Insipidus

doi: 10.3390/life14081012

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: After being blocked with 5% skim milk in PBS-T (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h, the membranes were probed overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-AQP2 (#AQP-002, Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-pSer256-AQP2 (#ab111346, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-CREB-1 (#06-863, Sigma-Aldrich), mouse monoclonal anti-p-CREB-1 (#sc-81486, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-β-actin (#A5441, Sigma-Aldrich).

Techniques:

Journal: Life

Article Title: Duloxetine-Induced Antidiuresis in Rats with Lithium-Induced Nephrogenic Diabetes Insipidus

doi: 10.3390/life14081012

Figure Lengend Snippet: Effects of duloxetine treatment on aquaporin-2 (AQP2) protein expression in lithium-induced nephrogenic diabetes insipidus. Immunoblots of AQP2 and pS256-AQP2 are shown; each lane was loaded with a protein sample from a different rat kidney ( A ). Densitometric analysis revealed that the protein levels of AQP2 and pS256-AQP2 were significantly reduced by lithium treatment (Li) compared to controls, but were restored by lithium/duloxetine co-treatment (Li + DX). The levels of AQP2 and pS256-AQP2 proteins increased in normal rats treated with duloxetine (DX) compared to the control group ( B ). Immunohistochemistry is shown for AQP2 ( C ) and pS256-AQP2 ( D ) in rat kidneys. Along the cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD), differences in AQP2 labeling among groups were consistent with those in the immunoblot analyses. Each bar in the densitometry results represents the mean ± standard deviation ( B ). *, p < 0.05 vs. control; # , p < 0.05 vs. Li by the post hoc Mann–Whitney U test. Scale bars, 50 μm.

Article Snippet: After being blocked with 5% skim milk in PBS-T (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h, the membranes were probed overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-AQP2 (#AQP-002, Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-pSer256-AQP2 (#ab111346, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-CREB-1 (#06-863, Sigma-Aldrich), mouse monoclonal anti-p-CREB-1 (#sc-81486, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-β-actin (#A5441, Sigma-Aldrich).

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Labeling, Standard Deviation, MANN-WHITNEY

Journal: Life

Article Title: Duloxetine-Induced Antidiuresis in Rats with Lithium-Induced Nephrogenic Diabetes Insipidus

doi: 10.3390/life14081012

Figure Lengend Snippet: Effects of duloxetine treatment on mRNA levels of aquaporin-2 (AQP2) and vasopressin-2 receptor (V2R) in lithium-induced nephrogenic diabetes insipidus. Quantitative PCR analyses of AQP2 ( A ) and V2R ( B ) were performed using whole kidneys. Compared with controls, the mRNA levels of AQP2 and V2R were decreased by lithium treatment (Li) and increased by lithium/duloxetine co-treatment (Li + DX). Each bar represents the mean ± standard deviation. *, p < 0.05 vs. control; # , p < 0.05 vs. Li by the post hoc Mann–Whitney U test.

Article Snippet: After being blocked with 5% skim milk in PBS-T (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h, the membranes were probed overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-AQP2 (#AQP-002, Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-pSer256-AQP2 (#ab111346, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-CREB-1 (#06-863, Sigma-Aldrich), mouse monoclonal anti-p-CREB-1 (#sc-81486, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-β-actin (#A5441, Sigma-Aldrich).

Techniques: Real-time Polymerase Chain Reaction, Standard Deviation, Control, MANN-WHITNEY

Journal: Life

Article Title: Duloxetine-Induced Antidiuresis in Rats with Lithium-Induced Nephrogenic Diabetes Insipidus

doi: 10.3390/life14081012

Figure Lengend Snippet: Reversal of duloxetine effects by tolvaptan co-treatment in lithium-induced nephrogenic diabetes insipidus. Immunoblots of AQP2, pS256-AQP2, CREB-1, and pCREB-1 are shown from the renal cortex ( A ) and medulla ( B ); each lane was loaded with a protein sample from a different rat kidney. Densitometric analysis revealed that the protein levels of AQP2, pS256-AQP2, and pCREB-1 were significantly reduced by lithium treatment (Li) compared to controls, but were restored by lithium/duloxetine co-treatment (Li + DX). However, all changes in both the cortex ( C ) and medulla ( D ) were reversed by the addition of tolvaptan (Li + DX + TV). Each group contained six rats, and the densitometry results are presented as the mean ± standard deviation. *, p < 0.05 vs. control; # , p < 0.05 vs. Li; † , p < 0.05 vs. Li + DX by the post hoc Mann–Whitney U test.

Article Snippet: After being blocked with 5% skim milk in PBS-T (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h, the membranes were probed overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-AQP2 (#AQP-002, Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-pSer256-AQP2 (#ab111346, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-CREB-1 (#06-863, Sigma-Aldrich), mouse monoclonal anti-p-CREB-1 (#sc-81486, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-β-actin (#A5441, Sigma-Aldrich).

Techniques: Western Blot, Standard Deviation, Control, MANN-WHITNEY

Journal: Biology Open

Article Title: Ammonia transport mediated by urea transporter A isoforms

doi: 10.1242/bio.061655

Figure Lengend Snippet: Summary of changes in surface pH following ammonia exposure of oocytes expressing wild-type or mutant mUT-A2, mUT-A3 , or mUT-B. (A) Changes in surface pH [ΔpH S(NH3) ] of oocytes expressing wild-type mUT-A2, mUT-A3 , or mUT-B following exposure to 0.5 M NH 3 /NH 4 + . (B) Changes in surface pH [ΔpH S(NH3) ] of water-injected oocytes following exposure to 0.5 M NH 3 /NH 4 + . (C) Surface pH measurements with water-injected ( n =10), water-injected with phloretin ( n =3), UT-A2 WT ( n =6), UT-A2 with phloretin ( n =5), UT-A2 T176V ( n =5), and UT-A2 T338V ( n =6) oocytes following exposure to 0.5 M NH 3 /NH 4 + . (D) Surface pH measurements with water-injected ( n =10), water-injected with phloretin ( n =3), UT-A3 WT ( n =3), UT-A3 with phloretin ( n =3), UT-A3 T246V ( n =4), and UT-A3 T408V ( n =4) oocytes following exposure to 0.5 M NH 3 /NH 4 + . (E) Surface pH measurements with water-injected ( n =10), water-injected with phloretin ( n =3), UT-B WT ( n =4), UT-B WT with phloretin ( n =4), UT-B T172V ( n =4), and UT-B T334V ( n =4) oocytes. Panels F–H on the right display background-subtracted results that yield channel-dependent activity. Oocytes expressing RhCG ( n =5; panels I and K) or AQP2 ( n =7; panels J and L) were used as positive and negative controls for assessing NH 3 transport. Gray circles indicate the values of each oocyte. Colored circles and triangles represent the averages of experiments performed on different days. The horizontal and vertical (error bars) lines on each plot represent the overall mean and the standard error of the mean of each group and condition. A standard two-tailed Student's t -test with Bonferroni correction was used to compare the difference between two means, as indicated above each graph. The significance level was set at P <0.0125.

Article Snippet: Experiments using oocytes injected with hAQP2 or hRhCG employed the same processing and detection protocols used for the UTs, except that a polyclonal anti-AQP2 antibody (Alpha Diagnostics, San Antonio, TX, USA) or a polyclonal antibody raised against the C-terminal region of RhCG ( ) and a goat anti-rabbit secondary antibody conjugated to HRP (AP132P; Millipore, Billerica, MA, USA) were used as reported previously ( , ).

Techniques: Expressing, Mutagenesis, Injection, Activity Assay, Two Tailed Test

Journal: Kidney Diseases

Article Title: Loss of Pten in Renal Tubular Cells Leads to Water Retention by Upregulating AQP2

doi: 10.1159/000528010

Figure Lengend Snippet: AQP2 was upregulated and concentrated in the apical plasma membrane of collecting ducts cells in the kidney of Pten Δ/Δ mice. a The expression of Aqp2 mRNA in the kidneys of Pten fl/fl and Pten Δ/Δ mice. b Western blots analysis of AQP2 and pS256-AQP2 levels in the kidney medulla lysates of Pten fl/fl and Pten Δ/Δ mice. c IHC staining of AQP2 in collecting ducts of Pten fl/fl and Pten Δ/Δ mice. Bars = 100 μm. d Higher magnification of IHC staining showed different distribution pattern of AQP2 in the collecting ducts cells of Pten fl/fl and Pten Δ/Δ mice. e Immunofluorescence staining of AQP2 (green) in collecting ducts of Pten fl/fl and Pten Δ/Δ mice. Higher magnifications of the selected areas are shown on the right.

Article Snippet: After blocked in 5% skim milk, the membranes were incubated with primary antibodies: anti-PTEN (9188L, CST, 1:2,000), anti-AQP2 (AQP-002, Alomone, 1:2,000), anti-pSer256-AQP2 (bs-12507R, Bioss, 1:500), anti-AKT (4691S, CST, 1:2,000), anti-pSer473-AKT (4060S, CST, 1:2,000), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000) at 4°C overnight.

Techniques: Membrane, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: Kidney Diseases

Article Title: Loss of Pten in Renal Tubular Cells Leads to Water Retention by Upregulating AQP2

doi: 10.1159/000528010

Figure Lengend Snippet: PTEN regulated AQP2 by dephosphorylating p-AKT. a IMCD cells were transfected with control shRNA (shControl) or Pten -specific shRNA (shPTEN), respectively. Relative expression level of Aqp2 genes in the shPTEN group is higher than that in the shControl group. b Western blots analysis of PTEN, AQP2, and p-AQP2 protein levels in shControl and shPTEN groups. c Western blots analysis of AKT and p-AKT proteins in kidney medulla lysates of Pten fl/fl and Pten Δ/Δ mice ( n = 3 for each group). IMCD3-shPTEN cells were treated with LY ( d ) or MK ( f ) for 24 h and cell lysates of different groups were subjected to immunoblotting to detect the protein levels of AKT, p-AKT, AQP2, and p-AQP2 ( n = 3-4 for each group). e, g Expression of membrane AQP2 (m-AQP2) and p-AQP2 (m-p-AQP2) in different groups of cells ( n = 3 for each group). ns p > 0.05, * p < 0.05, ** p < 0.02, *** p < 0.001.

Article Snippet: After blocked in 5% skim milk, the membranes were incubated with primary antibodies: anti-PTEN (9188L, CST, 1:2,000), anti-AQP2 (AQP-002, Alomone, 1:2,000), anti-pSer256-AQP2 (bs-12507R, Bioss, 1:500), anti-AKT (4691S, CST, 1:2,000), anti-pSer473-AKT (4060S, CST, 1:2,000), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000) at 4°C overnight.

Techniques: Transfection, Control, shRNA, Expressing, Western Blot, Membrane