primary antibodies aqp2  (Proteintech)

Journal: Journal of hypertension

Article Title: Collecting duct-specific knockout of sphingosine-1-phosphate receptor 1 aggravates DOCA-salt hypertension in mice

doi: 10.1097/HJH.0000000000002809

Figure Lengend Snippet: C onfirmation of S1PR1 knockout in the collecting duct. Upper: representative gel document of Western blot analysis showing S1PR1 protein levels in isolated medullary collecting tubules. Lower: representative photomicrographs showing double immunostaining of AQP2 (Green) and S1PR1 (Red).

Article Snippet: The kidney slides were stained using primary antibodies against AQP2 (rabbit polyclonal, Alomone, AQP-002) and S1PR1 (mouse monoclonal, EMD Millipore, MABC94), followed by incubation with either Alexa-488 or Alexa-555 labeled secondary antibody, and then examined with fluorescence microscopy (Olympus, Japan).

Techniques: Knock-Out, Western Blot, Isolation, Double Immunostaining

Journal: bioRxiv

Article Title: Loss of PRICKLE1 leads to abnormal endometrial epithelial architecture, decreased embryo implantation, and reduced fertility in mice

doi: 10.1101/2024.08.06.605120

Figure Lengend Snippet: (A) Prickle1 f/f targeting construct. (B) PCR genotyping of Prickle1 f/f mice (lanes 3 and 4), Prickle1 heterozygous mice (lane 1), and control mice (lanes 4, 5, and 6). (C,D) Representative images showing altered morphology of uterus in 6-month-old Prickle1 f/f Ltf +/icre cKO mouse (D) compared to control (C) mouse during diestrus. (E) Fertility assessment in control (n=14) and Prickle1 f/f Ltf +/icre cKO (n=23) 6-month-old mice showing decreased fertility in Prickle1 f/f Ltf +/icre cKO. (F) Implantation assessment at gestational day 4.5 of control (n=8) and Prickle1 f/f Ltf +/icre cKO (n=13) 6-month-old mice indicated two distinct groups of mutants. **P<0.05, ****P<0.0001. (G-J) Representative images showing implantation sites with corresponding H&E stain in 6-month-old Prickle1 f/f Ltf +/icre cKO mouse (H and J) compared to control (G and I) mouse at gestational day 4.5 showing abnormal fluid accumulation at proposed embryo site of Prickle1 f/f Ltf +/icre cKO. (Scale bar, 100 μm) (K,L) Optical z slice showing one horn of Prickle1 f/f Ltf +/icre cKO (L) and control uteri (K) with gestational day 4.5 embryos (*) with corresponding 3-dimensional surface models showing aberrant luminal folding in the mutant with no embryos present. (Scale bar, 500 μm) (PIR, peri-implantation region; IIR, inter-implantation region; IC, implantation chamber; M, mesometrial pole; AM, anti-mesometrial pole; O, ovary; Cx, cervix). (M) Uterine cross-sections of 6-month-old Prickle1 f/f Ltf +/icre cKO and control mice in diestrus stained with AQP2 (green) and DAPI (blue). Arrows indicate areas of low AQP2 expression. (Scale bar, 50 μm).

Article Snippet: For immunofluorescence staining, rehydrated tissue sections were stained as previously described ( ) using primary antibodies AQP2 (Novus Biologicals, Cat# NB110-74682SS, 1:100), E-CAD (Cell Signaling, Cat# 24E10, 1:100), ZEB1 (Cell Signaling, Cat# 70512, 1:400), TWIST1 (Cell Signaling, Cat# 90445, 1:100), and SNAI2 (Cell Signaling, Cat# 9585, 1:100).

Techniques: Construct, Control, Staining, Mutagenesis, Expressing

Journal: Kidney Diseases

Article Title: Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

doi: 10.1159/000538107

Figure Lengend Snippet: TGR5 activation increases the expression of AQP2 and AQP3 in the renal inner medulla. Renal medullary RNA and protein were extracted after LCA and INT-777 treatment for 3 days. a Quantitative PCR analysis showing the mRNA levels of AQP2, AQP3, and AQP4 in the renal medulla. b Western blot analysis showing the protein levels of AQP2, AQP3, and AQP4 in Control, LCA, and INT-777 groups. c Semi-quantification of western blot assays. Data represent the mean ± SEM. * p < 0.05, ** p < 0.01 compared to the Control group, n = 4. Note: Little effect of LCA and INT-777 on AQP4 mRNA and protein expression was observed.

Article Snippet: Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Kidney Diseases

Article Title: Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

doi: 10.1159/000538107

Figure Lengend Snippet: TGR5 activation by LCA and INT-777 induces AQP2 and AQP3 protein membrane translocation in mouse renal inner medullary collecting ducts. a Immunohistochemical assay showing that treatment of mice with LCA and INT-777 markedly increased AQP2 and AQP3 protein expression and their membrane translocation renal inner medullary. b Immunofluorescence study showing that treatment of mice with LCA and INT-777 markedly increased AQP2 and AQP3 protein expression and their membrane translocation in renal inner medullary. c , d Semi-quantification of mean integrated optical density of AQP2 and AQP3 in ( a ). e , f Semi-quantification of mean integrated optical density of AQP2 and AQP3 in ( b ). Note: Apical AQP2 protein expression and basolateral AQP3 protein expression were dramatically increased after LCA and INT-777 treatment.

Article Snippet: Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004).

Techniques: Activation Assay, Membrane, Translocation Assay, Immunohistochemical staining, Expressing, Immunofluorescence

Journal: Kidney Diseases

Article Title: Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

doi: 10.1159/000538107

Figure Lengend Snippet: LCA and INT-777 promote the expression of AQP2 and AQP3 in a TGR5-dependent manner. Rat IMCDs were transfected with TGR5 siRNA or negative control for 36 h and then stimulated with 5 μ m LCA and INT-777 for 12 h. The mRNA expression of AQP2, AQP3, and AQP4 was measured by quantitative PCR. a–c Knockdown of TGR5 abolished the effect of LCA on AQP2 and AQP3 expression. LCA treatment significantly upregulated AQP2 ( a ) and AQP3 ( b ) mRNA expression, with little effect on AQP4 ( c ). LCA-induced AQP2 and AQP3 mRNA expression were completely abolished by the knockdown of TGR5. d–f Knockdown of TGR5 abolished the effect of INT-777 on AQP2 and AQP3 expression. INT-777 treatment significantly upregulated AQP2 ( d ) and AQP3 ( e ) mRNA expression, with little effect on AQP4 ( f ). INT-777-induced AQP2 and AQP3 mRNA expression were completely abolished by the knockdown of TGR5. g , h The protein levels of AQP2, AQP3, and AQP4 were measured by Western blot assay. LCA ( g ) and INT-777 ( h ) treatment induced AQP2 and AQP3 protein expression, which was markedly attenuated by the knockdown of TGR5. Data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the Control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the LCA or INT-777 group, n = 3.

Article Snippet: Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004).

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

Journal: Kidney Diseases

Article Title: Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

doi: 10.1159/000538107

Figure Lengend Snippet: TGR5 activation induces AQP2 and AQP3 mRNA expression via a PKA-dependent manner in primary cultured rat IMCDs. Rat IMCDs were pretreated with vehicle or H89 (10 μ m ) for 1 h and then stimulated with 5 μ m LCA and INT-777 for 12 h. The mRNA expression of AQP2, AQP3, and AQP4 was measured by quantitative PCR. a–c H89 treatment abolished the effect of LCA on AQP2 and AQP3 expression. LCA treatment significantly upregulated AQP2 ( a ) and AQP3 ( b ) mRNA expression, with little effect on AQP4 ( c ). LCA-induced AQP2 and AQP3 mRNA expression were completely abolished by H89 treatment. d–f H89 treatment abolished the effect of INT-777 on AQP2 and AQP3 expression. INT-777 treatment significantly upregulated AQP2 ( d ) and AQP3 ( e ) mRNA expression, with little effect on AQP4 ( f ). INT-777-induced AQP2 and AQP3 mRNA expression were completely abolished by H89 treatment. Data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the Control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the LCA or INT-777 group, n = 3.

Article Snippet: Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004).

Techniques: Activation Assay, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Control

Journal: Kidney Diseases

Article Title: Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

doi: 10.1159/000538107

Figure Lengend Snippet: Effect of H89 on LCA- and INT-777-induced AQP2 and AQP3 protein expression and the phosphorylation and nuclear translocation of CREB protein in primary cultured IMCDs Rat IMCDs were pretreated with DMSO (vehicle) or H89 (10 μ m ) for 1 h and then stimulated with LCA (5 μ m ) or INT-777 (5 μ m ) for 12 h. a , b Western blot assay showing that LCA ( a ) and INT-777 ( b ) treatment markedly induced AQP2 and AQP3 protein expression and the phosphorylation and nuclear translocation of CREB protein levels. c , d Immunofluorescence analysis demonstrating that LCA and INT-777 promoted nuclear translocation of CREB, which was markedly attenuated by the pretreatment with H89.

Article Snippet: Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004).

Techniques: Expressing, Phospho-proteomics, Translocation Assay, Cell Culture, Western Blot, Immunofluorescence

Journal: Kidney Diseases

Article Title: Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

doi: 10.1159/000538107

Figure Lengend Snippet: CREB transcriptionally induces AQP2 and AQP3 gene expression. a , b Schematic demonstration of the location of a putative CREB-binding element located in the mouse AQP2 ( a ) and AQP3 ( b ) gene promoter. c , d Luciferase reporter assay showing that CREB overexpression significantly increased the promoter activity of AQP2 gene ( c ) and AQP3 gene ( d ). **** p < 0.0001, n = 8. e , f Gel EMSA demonstrating that CREB can directly bind to the predicted CREB-binding site located in the promoter region of AQP2 ( e ) and AQP3 ( f ) gene. Note: Excessive unlabeled probe markedly attenuated the binding of CREB protein to the labeled AQP2 and AQP3 promoter probe.

Article Snippet: Immunostaining was performed overnight at 4°C using the following primary antibodies against AQP2 (ab199975), AQP3 (ab125219), AQP4 (sc-32739), p-CREB (CST#9198), CREB (CST#9197), and β-actin (Abclonal #AC004).

Techniques: Gene Expression, Binding Assay, Luciferase, Reporter Assay, Over Expression, Activity Assay, Labeling