rabbit polyclonal anti aqp5 antibody  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

doi: 10.3390/ijms22020619

Figure Lengend Snippet: Spatiotemporal expression of CXC-chemokine receptor 4 (CXCR4) and its ligand, CXCL12, and developmental genes in embryonic organs. ( A ) Schematic diagram of embryonic submandibular gland (eSMG) isolation and ex vivo culture. ( B ) Ex vivo branching morphogenesis of eSMGs from embryonic day (E) 13 to 17, showing epithelial growth and retraction of mesenchyme. Scale bars: 500 µm. ( C ) Temporal mRNA expression patterns of keratin 7 ( Krt7 ), aquaporin 5 ( Aqp5 ), e-cadherin ( Cdh1 ), Krt15 , Cxcr4 , and Cxcl12 were measured from E13 to E17 by qPCR ( n = 3). ( D ) Epithelial (Epi) and mesenchymal (Mes) expression of Cxcr4 , Cxcl12 , odd-skipped related transcription factor 1 ( Osr1 ), and Cdh1 were quantified by qPCR at E13. The comparative C t values are expressed as fold increase relative to the epithelium ( n = 3). ( E ) Representative images showing expression of CXCR4 and CXCL12 in eSMG (upper) and their colocalization (lower) ( n = 3, scale bar: 500 µm). ( F ) Representative immunofluorescence images of CXCR4 and CXCL12 expression in E12 embryonic lung and pancreas ( n = 4); whole view (left two panels; scale bar: 500 µm) and magnified lumen structures (right two panels; scale bar: 50 µm). Data are presented as the mean ± SEM; * p

Article Snippet: The following primary antibodies were used in the procedures: rat monoclonal anti-CXCR4 antibody (R & D Systems, MAB21651), rabbit monoclonal anti-KRT7 antibody (Abcam, ab181598; Cambridge, UK); rabbit monoclonal anti-CDH1 antibody (CST, 3195; Beverly, MA, USA), rabbit monoclonal anti-H2AK119ub antibody (CST, 8240); mouse monoclonal anti-CXCL12 antibody (Novus Biologicals, MAB350; Littleton, CO, USA), rabbit polyclonal anti-AQP5 antibody (Alomone Labs, AQP-005; Jerusalem, Israel), rabbit monoclonal anti-CASP3 antibody (CST, 9664), and rat monoclonal anti-KI67 antibody (ThermoFisher, 14-5698-82).

Techniques: Expressing, Isolation, Ex Vivo, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

doi: 10.3390/ijms22020619

Figure Lengend Snippet: AMD3100-induced precocious differentiation of epithelial cells. ( A , B ) Representative contour tracing ( A ) and bud number changes ( B ) of control and AMD3100-treated eSMGs during 48 h at 6-h intervals ( n = 3). ( C ) EdU staining results at 6 and 24 h after AMD3100 treatment. EdU in green and PNA in gray ( n = 4, scale bar: 500 µm). ( D ) Immunostaining results of Ki67 (red) and F-actin (green) in acini and duct of eSMGs 24 h after AMD3100 treatment. Morphologies of acinar buds and duct cells are outlined with white dotted lines. Scale bar: 50 µm. ( E ) Duct widths of control and AMD3100-treated eSMGs were visualized via F-actin-based intensity profiles of horizontal sectioning of ducts 24 h after the treatment. ( F ) Duct widths of control and AMD3100-treated eSMGs were quantified 24 h after the treatment ( n = 9). ( G ) Immunostaining results of AQP5 (green) and KRT7 (red). Magnified regions of acinar buds are marked with white dotted squares. The white arrows (middle panels) indicate areas with the highest AQP5 expression ( n = 4, scale bar: left, 100 µm; middle and right, 50 µm). Data are presented as the mean ± SEM; * p

Article Snippet: The following primary antibodies were used in the procedures: rat monoclonal anti-CXCR4 antibody (R & D Systems, MAB21651), rabbit monoclonal anti-KRT7 antibody (Abcam, ab181598; Cambridge, UK); rabbit monoclonal anti-CDH1 antibody (CST, 3195; Beverly, MA, USA), rabbit monoclonal anti-H2AK119ub antibody (CST, 8240); mouse monoclonal anti-CXCL12 antibody (Novus Biologicals, MAB350; Littleton, CO, USA), rabbit polyclonal anti-AQP5 antibody (Alomone Labs, AQP-005; Jerusalem, Israel), rabbit monoclonal anti-CASP3 antibody (CST, 9664), and rat monoclonal anti-KI67 antibody (ThermoFisher, 14-5698-82).

Techniques: Staining, Immunostaining, Expressing

Journal: Biomaterials

Article Title: Neurturin-containing laminin matrices support innervated branching epithelium from adult epithelial salispheres

doi: 10.1016/j.biomaterials.2019.119245

Figure Lengend Snippet: Innervated salisphere-derived branching epithelial structures resemble an ex vivo fetal gland. D4 recombined mouse adult salispheres (A) and fetal epithelium (B) in laminin hydrogels with Mes, PSG and NRTN were stained for Peanut Agglutinin (PNA, red) to outline the epithelia, TUBB3 (green) for nerves, K5 (cyan), KIT (cyan), AQP5 (red), KI67 (green) for proliferation and/or DAPI (blue) for nuclei. Confocal images of 10μm sections. Scale bars, 100 μm (A) and 50 μm (B) .

Article Snippet: Samples were blocked for 90 minutes with 10% donkey serum (Jackson Laboratories, ME), 1% BSA, and MOM IgG blocking-reagent (Vector Laboratories, CA) in 0.1% PBS-Tween-20, and incubated with primary-antibodies overnight at 4°C, including rat-anti-Kit (1:100, R & D Systems, MN), rabbit-anti-Keratin-5 (1:2000, Covance Research, NJ), rat-anti-Keratin-19 (1:300, Developmental Biology Hybridoma Bank, University of Iowa) ), rabbit-Aqp5 (1:200, Alomone Labs, Israel), mouse-anti-SMA (1:200, Sigma Aldrich, MO), mouse-anti-Ki67 (BD Biosciences), rabbit-anti-Keratin-14 (1:2000, Covance Research, NJ) and rabbit-anti-E-cadherin (1:100,Cell Signaling Technology, MA).

Techniques: Derivative Assay, Ex Vivo, Staining

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell–Specific Genes

doi: 10.1165/rcmb.2016-0071OC

Figure Lengend Snippet: Expression of known AT1 cell markers in rat microarray data. Log2 expression data were generated from microarray experiments: AT1-like cells differentiated in culture ( blue ) (Day 2-6), freshly isolated AT1 cells ( purple ), freshly isolated AT2 cells ( red ), and other tissues ( black ). Table at bottom right includes previously described AT1 cell–specific genes, their associated Illumina Probe IDs, and their FDR-corrected P values in this study. FDR adjustment is based on the number of tests shown for known genes. AGER, advanced glycosylation end product–specific receptor; AQP5, aquaporin 5; CAV, caveolin; FDR, false-discovery rate; ILMN, Illumina probe number; PDPN, podoplanin. **Indicates significantly greater in rat AT1 and AT1-like cells compared to all others.

Article Snippet: Blots were incubated with rabbit anti–epithelial sodium channel (ENaC) γ (1:200, sc-21014; Santa Cruz Biotechnology, Santa Cruz, CA), anti-semaphorin 3B (SEMA3B) (1:500, ; Abnova, Jhongli, Taiwan), anti-SEMA3E (1:100, AP7976b; Abgent, San Diego, CA), anti-GRAMD2 (1:100, ab84567; Abcam, Cambridge, MA), anti-SFTPC (1:200, AB3786; Millipore, Billerica, MA), and anti-AQP5 (1:200, AQP-005; Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Microarray, Generated, Isolation

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell–Specific Genes

doi: 10.1165/rcmb.2016-0071OC

Figure Lengend Snippet: Validation of GRAMD2 expression in AT1 cells in lung tissue. ( A ) Confocal images for GRAMD2/SFTPC double staining in mouse lung sections show that GRAMD2 does not colocalize with SFTPC. DAPI is the nuclear counterstain. Scale bar : 20 µm. ( B ) Confocal images for GRAMD2/AQP5 double staining in mouse lung sections shows that GRAMD2 colocalizes with AQP5. DAPI is the nuclear counterstain. Scale bar : 20 µm. DAPI, 4’,6-diamidino-2-phenylindole.

Article Snippet: Blots were incubated with rabbit anti–epithelial sodium channel (ENaC) γ (1:200, sc-21014; Santa Cruz Biotechnology, Santa Cruz, CA), anti-semaphorin 3B (SEMA3B) (1:500, ; Abnova, Jhongli, Taiwan), anti-SEMA3E (1:100, AP7976b; Abgent, San Diego, CA), anti-GRAMD2 (1:100, ab84567; Abcam, Cambridge, MA), anti-SFTPC (1:200, AB3786; Millipore, Billerica, MA), and anti-AQP5 (1:200, AQP-005; Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Double Staining

Journal: PLoS ONE

Article Title: Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

doi: 10.1371/journal.pone.0026956

Figure Lengend Snippet: AdHSP reduces the abundance of caspases 8 and 9 in 2CLP-induced lung injury. A. Immunoblotting of whole cytosolic extracts for Caspase 8 and 9. Abbreviations as in Fig. 1 . Upper panel : Representative autoradiogram of SDS-PAGE, 30 µg of cytosolic extract/lane. Primary rabbit polyclonal antibody to caspase 8. Secondary goat anti rabbit IgG. .Graphic representation of relative density of cytosolic caspase 8. Middle panel : Representative autoradiogram of SDS-PAGE, 30 µg of cytosolic extracts. Primary rabbit polyclonal antibody to caspase 9, secondary goat anti rabbit IgG. Graph - Graphic representation of relative density (mean +/− standard deviation) of cytosolic caspase 9. * = significantly different from 2CLPPBS and 2CLPAdGFP. Lower panels: Representative autoradiogram of SDS-PAGE, 30 µg of mitochondrial extract/lane. Primary mouse monoclonal antibody to Bcl2, secondary goat anti mouse IgG and primary mouse monoclonal antibody to COX IV, secondary goat anti mouse IgG. COX IV serves as mitochondrial loading control. B. Hsp70 in vivo interaction with apopotosomal Apaf-1. Representative autoradiograms. Samples were immunoprecipitated with a rabbit polyclonal antibody to Apaf-1 and subjected to SDS-PAGE. Upper panels: Immunoblotting with a primary rabbit polyclonal antibody to pro-caspase 9, secondary goat anti rabbit IgG. Middle panels: Immunoblotting with primary mouse monoclonal antibody to Hsp70, secondary goat anti mouse IgG. Lower panels: IgG detection IgG serves as loading control. 250 µg of cytosolic extracts obtained from TO, 2CLPPBS, 2CLPAdHSP or 2CLPAdGFP treated animals sacrificed 48 hrs after the induction of sepsis. C. Hsp70 in vitro, MLE-12 cells, interaction with apopotosomal Apaf-1. Representative autoradiograms. 250 µg of cytosolic extracts obtained from non treated MLE-12 cells (controls), stimulated with tumor necrosis factor (TNF) and treated with AdHSP or AdGFP. Samples were immunoprecipitated with a rabbit polyclonal antibody to Apaf-1 and subjected to SDS-PAGE. Upper panels: Immunoblotting with a primary rabbit polyclonal antibody to pro-caspase 9, secondary goat anti rabbit IgG. Middle panels: Immunoblotting with primary mouse monoclonal antibody to Hsp70, secondary goat anti mouse IgG. Lower panels: IgG serves as loading control. D. Apaf-1 – CARD dissociates from Pro-caspase-9 in the presence of Hsp70. Representative autoradiograms. 100 µg of cytosolic extracts obtained from 2CLPPBS and 2CLAdHSP treated animals, were immunoprecipitated with Pro-caspase-9 and further incubated with GST-Apaf-1 – CARD obtained from BL-21 cells, together with 5 mM ATP and 5 µg/ml human Cytochrome C for 5, 10, 20 and 30 minutes. Samples were subjected to SDS-PAGE, immunoblotted and the membranes were incubated with primary rabbit polyclonal antibody to Cleaved Caspase-9, secondary to goat anti rabbit IgG.

Article Snippet: AQP5 was detected using a primary rabbit polyclonal antibody (Almone Inc., Jerusalem, Israel) diluted 1∶100 and a secondary anti rabbit IgG.

Techniques: SDS Page, Standard Deviation, In Vivo, Immunoprecipitation, In Vitro, Incubation

Journal: PLoS ONE

Article Title: Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

doi: 10.1371/journal.pone.0026956

Figure Lengend Snippet: AdHSP alters interactions between Caspase 8, 9, 3 and Apaf-1. A and B: Representative Autoradiogram Demonstrating AdHSP treatment disrupts interaction between caspase 8 and caspase 9. Representative autoradiograms. 250 µg of cytosolic extracts were immunoprecipitated with rabbit polyclonal antibody to caspase 8 or caspase-9 and subjected to SDS-PAGE. Immunoblotting performed with either primary rabbit polyclonal antibodies to caspase 9 or caspase-8, secondary goat anti rabbit IgG. Lower panels: IgG detection. IgG serves as loading control. Abbreviations as in Figure 1 . C: Hsp70 disrupts caspases 3, 8 9 and Apaf-1 complexes. 250 µg of cytosolic extracts from lung tissue fractionated via column chromatography, eluted by molecular weight, immunoprecipitated with an antibody to caspase-9 and subjected to 9% SDS-PAGE. Molecular weight of each fraction (kDa) indicated at the top of the figure. Detecting antibodies (anti-caspase 9, anti-caspase 8, anti-pro-caspase 3, anti-Hsp70 and anti-Apaf-1,) noted to the left of the panels. Lower panel: IgG detection. IgG serves as loading control. Abbreviations as in Figure 1 .

Article Snippet: AQP5 was detected using a primary rabbit polyclonal antibody (Almone Inc., Jerusalem, Israel) diluted 1∶100 and a secondary anti rabbit IgG.

Techniques: Immunoprecipitation, SDS Page, Column Chromatography, Molecular Weight

Journal: PLoS ONE

Article Title: Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

doi: 10.1371/journal.pone.0026956

Figure Lengend Snippet: AdHSP prevents nuclear translocation of activated Caspase-3. A. Representative autoradiograms for pro-caspase 3 and activated (cleaved) Caspase 3. 30 µg of cytosolic (upper panels) and nuclear (lower panel) extracts were subjected to SDS-PAGE. Immunoblotting performed with primary rabbit polyclonal antibody to pro- caspase 3 and secondary goat anti rabbit IgG, primary goat antibody to β-actin and secondary donkey anti goat IgG, primary rabbit polyclonal antibody to active (cleaved) caspase-3 and secondary goat anti rabbit IgG, primary mouse monoclonal antibody to histone (H1) and secondary goat anti mouse IgG.. β-actin and histone serve as loading controls. B. Representative stained fixed tissue section depicting intra-nuclear staining for Caspase 3. Sections obtained from T0 control, 2CLPPBS and 2CLPHSP rats. Tissue isolated 48 hrs after the induction of sepsis. Upper panel : 40× magnifications. Black arrows indicate active caspase 3 stained nuclei. Lower panel : 100× magnification of upper panel. C. Caspase 3 Activity Assay. Graphic representation of relative caspase-3 enzymatic activity (mean +/− standard deviation) * = significantly different from Control and AdHSP+TNF.

Article Snippet: AQP5 was detected using a primary rabbit polyclonal antibody (Almone Inc., Jerusalem, Israel) diluted 1∶100 and a secondary anti rabbit IgG.

Techniques: Translocation Assay, SDS Page, Staining, Isolation, Caspase-3 Activity Assay, Activity Assay, Standard Deviation