nr2a  (Alomone Labs)


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    Structured Review

    Alomone Labs nr2a
    CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, <t>NR2A,</t> NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p
    Nr2a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo"

    Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

    Journal: eNeuro

    doi: 10.1523/ENEURO.0175-17.2017

    CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p
    Figure Legend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

    Techniques Used: Infection, Western Blot

    2) Product Images from "Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses"

    Article Title: Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses

    Journal: The EMBO Journal

    doi: 10.1002/embj.201386356

    Synaptic GluN2B-NMDAR are laterally redistributed in the synaptic area following chem LTP in immature neurons Detection of a single QD (30-Hz acquisition) in our experimental conditions. The high signal-to-noise ratio (SNR) ( > 5) enables the detection and location of the signal with a high pointing accuracy (˜30 nm). The QD fluorescence is quantified on a pseudocolor scale (low: red; high: yellow). Scale bar = 800 nm. A 500-frame stack is obtained while tracking down a single NMDAR/QD complex. On each frame, a single GluN2B- (green) or GluN2A- (blue) QD particle complex is detected and precisely located within synaptic (dark gray) and perisynaptic (320-nm annulus around the synapse; light gray) areas. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex during the 500-frame stack. Note that GluN2A-NMDAR are more concentrated within the core of the PSD. Scale bar image = 300 nm; synaptic areas = 200 nm. Relative fraction of synaptic and perisynaptic GluN2-QD particles, calculated before and after chem LTP for both GluN2B- (left) and GluN2A-NMDAR (right) ( n = 25 and 20 dendritic fields before and after chem LTP, respectively). Note the significant decrease in the relative synaptic content in GluN2B-NMDAR particles right after chem LTP (Student's t -test, ** P
    Figure Legend Snippet: Synaptic GluN2B-NMDAR are laterally redistributed in the synaptic area following chem LTP in immature neurons Detection of a single QD (30-Hz acquisition) in our experimental conditions. The high signal-to-noise ratio (SNR) ( > 5) enables the detection and location of the signal with a high pointing accuracy (˜30 nm). The QD fluorescence is quantified on a pseudocolor scale (low: red; high: yellow). Scale bar = 800 nm. A 500-frame stack is obtained while tracking down a single NMDAR/QD complex. On each frame, a single GluN2B- (green) or GluN2A- (blue) QD particle complex is detected and precisely located within synaptic (dark gray) and perisynaptic (320-nm annulus around the synapse; light gray) areas. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex during the 500-frame stack. Note that GluN2A-NMDAR are more concentrated within the core of the PSD. Scale bar image = 300 nm; synaptic areas = 200 nm. Relative fraction of synaptic and perisynaptic GluN2-QD particles, calculated before and after chem LTP for both GluN2B- (left) and GluN2A-NMDAR (right) ( n = 25 and 20 dendritic fields before and after chem LTP, respectively). Note the significant decrease in the relative synaptic content in GluN2B-NMDAR particles right after chem LTP (Student's t -test, ** P

    Techniques Used: Fluorescence

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    Alomone Labs anti p2x4 receptor antibody
    Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with <t>P2X4</t> receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.
    Anti P2x4 Receptor Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit anti aqp2 antibody
    Mass spectrometry analysis of synthetic peptides corresponding to the COOH-terminal tail of human <t>AQP2</t> did not detect any significant phosphorylation of the water channel by AMPK. Protease-treated peptides (trypsin or LysC) or full-length peptides are indicated with bold underlined amino acids, indicating where phosphorylation was detected. The no. in parentheses indicates the adjusted spectral count of that particular phosphorylation site. The adjusted spectral count is obtained by multiplying the spectral count of each site with its phosphorylated probability reported by PhosphoRS. The last column indicates the percent sequence coverage. SAMS peptide was used as a positive control.
    Rabbit Anti Aqp2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with P2X4 receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.

    Journal: International Journal of Molecular Sciences

    Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

    doi: 10.3390/ijms222010970

    Figure Lengend Snippet: Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with P2X4 receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.

    Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

    Techniques:

    Co-immunoprecipitation of MFG-E8 and P2X4. Representative Western blots showing co-immunoprecipitation of MFG-E8 and P2X4R proteins in human microglial cells treated (or not) with EVs for 2 h, + indicates treatment with appropriate antibody. Full blots are available in Supplementary Figure S4 .

    Journal: International Journal of Molecular Sciences

    Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

    doi: 10.3390/ijms222010970

    Figure Lengend Snippet: Co-immunoprecipitation of MFG-E8 and P2X4. Representative Western blots showing co-immunoprecipitation of MFG-E8 and P2X4R proteins in human microglial cells treated (or not) with EVs for 2 h, + indicates treatment with appropriate antibody. Full blots are available in Supplementary Figure S4 .

    Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

    Techniques: Immunoprecipitation, Western Blot

    Mass spectrometry analysis of synthetic peptides corresponding to the COOH-terminal tail of human AQP2 did not detect any significant phosphorylation of the water channel by AMPK. Protease-treated peptides (trypsin or LysC) or full-length peptides are indicated with bold underlined amino acids, indicating where phosphorylation was detected. The no. in parentheses indicates the adjusted spectral count of that particular phosphorylation site. The adjusted spectral count is obtained by multiplying the spectral count of each site with its phosphorylated probability reported by PhosphoRS. The last column indicates the percent sequence coverage. SAMS peptide was used as a positive control.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

    doi: 10.1152/ajprenal.00308.2016

    Figure Lengend Snippet: Mass spectrometry analysis of synthetic peptides corresponding to the COOH-terminal tail of human AQP2 did not detect any significant phosphorylation of the water channel by AMPK. Protease-treated peptides (trypsin or LysC) or full-length peptides are indicated with bold underlined amino acids, indicating where phosphorylation was detected. The no. in parentheses indicates the adjusted spectral count of that particular phosphorylation site. The adjusted spectral count is obtained by multiplying the spectral count of each site with its phosphorylated probability reported by PhosphoRS. The last column indicates the percent sequence coverage. SAMS peptide was used as a positive control.

    Article Snippet: The rabbit anti-AQP2 antibody was obtained from Alomone Labs (Jerusalem, Israel).

    Techniques: Mass Spectrometry, Sequencing, Positive Control

    AMPK activator AICAR prevents apical membrane accumulation of AQP2. Polarized mpkCCD c14 cells were treated with vehicle (DMSO) or the AMPK activator AICAR (1 mM) for 4 h and then 10 μM forskolin (vs. vehicle) for the last 2 h before cell harvesting. 5% of the cell lysates were used for immunoblotting using antibodies against AQP2 or actin as a loading control. The rest of the lysates were affinity purified using streptavidin followed by immunoblotting. A : representative immunoblots of 3 separate surface biotinylation experiments are shown. B : summary densitometric data comparing the mean (±SE) amounts of apical AQP2 as a percentage of that in the control condition for nonglycosylated (solid bars), core glycosylated (open bars), and fully glycosylated (shaded bars) forms of AQP2 are shown. Nonglycosylated: * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

    doi: 10.1152/ajprenal.00308.2016

    Figure Lengend Snippet: AMPK activator AICAR prevents apical membrane accumulation of AQP2. Polarized mpkCCD c14 cells were treated with vehicle (DMSO) or the AMPK activator AICAR (1 mM) for 4 h and then 10 μM forskolin (vs. vehicle) for the last 2 h before cell harvesting. 5% of the cell lysates were used for immunoblotting using antibodies against AQP2 or actin as a loading control. The rest of the lysates were affinity purified using streptavidin followed by immunoblotting. A : representative immunoblots of 3 separate surface biotinylation experiments are shown. B : summary densitometric data comparing the mean (±SE) amounts of apical AQP2 as a percentage of that in the control condition for nonglycosylated (solid bars), core glycosylated (open bars), and fully glycosylated (shaded bars) forms of AQP2 are shown. Nonglycosylated: * P

    Article Snippet: The rabbit anti-AQP2 antibody was obtained from Alomone Labs (Jerusalem, Israel).

    Techniques: Cell Harvesting, Affinity Purification, Western Blot

    AMPK activator AICAR induces intracellular redistribution of AQP2 in ex vivo kidney slices. A : confocal images of AQP2 immunofluorescence labeling (green) in kidney slices incubated in Ringer buffer alone for 75 min display an apical distribution of the water channel. Asterisks indicate the position of the lumen of the collecting ducts. B : addition of 1 mM AICAR for 75 min induced a cytosolic distribution of AQP2. C : incubation of kidney slices with dDAVP for the last 15 min of a 75-min incubation revealed apical accumulation of AQP2. D : however, preincubation of slices with AICAR did not prevent the dDAVP-induced apical accumulation of AQP2. E : regions of interest (ROIs; example shown in A ). Quantification of the mean (±SE) AQP2-associated mean pixel intensity (MPI) of apical-to-cytoplasmic ratio [arbitrary units (AU)] relative to that of cells measured under the control condition was used as a measure of apical AQP2 accumulation. Additional incubations of kidney slices in Ringer buffer were performed in the absence ( F ) or presence ( G ) of the PKA inhibitor myristoylated protein kinase inhibitor (mPKI; 10 μM) for 75 min. H : the relative AQP2 mean pixel intensity apical-to-cytoplasmic ratio was reduced dramatically with mPKI treatment. Data were obtained from at least 3 separate kidney slice experiments, using kidneys from at least 3 animals, and measuring a total of at least 30 cells per condition. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

    doi: 10.1152/ajprenal.00308.2016

    Figure Lengend Snippet: AMPK activator AICAR induces intracellular redistribution of AQP2 in ex vivo kidney slices. A : confocal images of AQP2 immunofluorescence labeling (green) in kidney slices incubated in Ringer buffer alone for 75 min display an apical distribution of the water channel. Asterisks indicate the position of the lumen of the collecting ducts. B : addition of 1 mM AICAR for 75 min induced a cytosolic distribution of AQP2. C : incubation of kidney slices with dDAVP for the last 15 min of a 75-min incubation revealed apical accumulation of AQP2. D : however, preincubation of slices with AICAR did not prevent the dDAVP-induced apical accumulation of AQP2. E : regions of interest (ROIs; example shown in A ). Quantification of the mean (±SE) AQP2-associated mean pixel intensity (MPI) of apical-to-cytoplasmic ratio [arbitrary units (AU)] relative to that of cells measured under the control condition was used as a measure of apical AQP2 accumulation. Additional incubations of kidney slices in Ringer buffer were performed in the absence ( F ) or presence ( G ) of the PKA inhibitor myristoylated protein kinase inhibitor (mPKI; 10 μM) for 75 min. H : the relative AQP2 mean pixel intensity apical-to-cytoplasmic ratio was reduced dramatically with mPKI treatment. Data were obtained from at least 3 separate kidney slice experiments, using kidneys from at least 3 animals, and measuring a total of at least 30 cells per condition. * P

    Article Snippet: The rabbit anti-AQP2 antibody was obtained from Alomone Labs (Jerusalem, Israel).

    Techniques: Ex Vivo, Immunofluorescence, Labeling, Incubation

    AMPK inhibition promotes AQP2-mediated oocyte swelling and shortens time to lysis. Oocytes were microinjected with cRNAs for AQP2 and the different indicated AMPK constructs [WT-AMPK-α (●), dominant-negative (DN) AMPK-α1-K45R (■), and constitutively active (CA) AMPK-γ1-R70Q (▲)]. After 3 days, the oocytes were subjected to hypotonic shock in deionized water. Oocyte times to lysis were assessed by microscopy, and the mean times to lysis (normalized to that of the WT-AMPK-expressing group for each batch) are shown for all of the data obtained for these 3 groups. Oocytes expressing DN-AMPK had a significantly reduced time to lysis relative to each of the other two groups. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

    doi: 10.1152/ajprenal.00308.2016

    Figure Lengend Snippet: AMPK inhibition promotes AQP2-mediated oocyte swelling and shortens time to lysis. Oocytes were microinjected with cRNAs for AQP2 and the different indicated AMPK constructs [WT-AMPK-α (●), dominant-negative (DN) AMPK-α1-K45R (■), and constitutively active (CA) AMPK-γ1-R70Q (▲)]. After 3 days, the oocytes were subjected to hypotonic shock in deionized water. Oocyte times to lysis were assessed by microscopy, and the mean times to lysis (normalized to that of the WT-AMPK-expressing group for each batch) are shown for all of the data obtained for these 3 groups. Oocytes expressing DN-AMPK had a significantly reduced time to lysis relative to each of the other two groups. * P

    Article Snippet: The rabbit anti-AQP2 antibody was obtained from Alomone Labs (Jerusalem, Israel).

    Techniques: Inhibition, Lysis, Construct, Dominant Negative Mutation, Microscopy, Expressing

    AMPK fails to significantly phosphorylate AQP2 in vitro. V5-tagged AQP2 was expressed in HEK-293 cells, immunoprecipitated, and incubated with [γ- 32 P]ATP in the presence of PKA catalytic subunit (positive control) or in the presence or absence of AMPK holoenzyme. A phosphoscreen image ( top ) and immunoblot ( bottom ) of the same membrane are shown (representative of 4 experiments). AQP2 gets robustly phosphorylated in the presence of PKA, but only weakly phosphorylated in the presence of AMPK ( top ). PKA catalytic subunit and the AMPK β-subunit get autophosphorylated, as indicated. The immunoblot ( bottom ) reveals similar AQP2 loading in all lanes.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

    doi: 10.1152/ajprenal.00308.2016

    Figure Lengend Snippet: AMPK fails to significantly phosphorylate AQP2 in vitro. V5-tagged AQP2 was expressed in HEK-293 cells, immunoprecipitated, and incubated with [γ- 32 P]ATP in the presence of PKA catalytic subunit (positive control) or in the presence or absence of AMPK holoenzyme. A phosphoscreen image ( top ) and immunoblot ( bottom ) of the same membrane are shown (representative of 4 experiments). AQP2 gets robustly phosphorylated in the presence of PKA, but only weakly phosphorylated in the presence of AMPK ( top ). PKA catalytic subunit and the AMPK β-subunit get autophosphorylated, as indicated. The immunoblot ( bottom ) reveals similar AQP2 loading in all lanes.

    Article Snippet: The rabbit anti-AQP2 antibody was obtained from Alomone Labs (Jerusalem, Israel).

    Techniques: In Vitro, Immunoprecipitation, Incubation, Positive Control

    Changes in AQP2 phosphorylation pattern following dDAVP treatment in the presence or absence of AMPK activator in mpkCCD c14 cells. Twenty-four hours posttransfection with wild-type AQP2 plasmid, cells were incubated in the presence or absence of AICAR (2 mM) for 4 h, and then dDAVP (10 nM) or vehicle was added for 30 min before cell harvesting. Immunoblot analysis of immunoprecipitated V5-AQP2 was performed using the corresponding rabbit polyclonal antibodies against phosphorylated AQP2 COOH-terminal sites: −S256, −S261, −S264, and −S269. To confirm successful immunoprecipitation, the membranes were reblotted using anti-AQP2 and anti-V5 antibodies coupled to HRP. A : representative set of immunoblots from these experiments using antibodies against AQP2 and its different COOH-terminal phosphorylation sites. B : quantification of AQP2 phosphorylation signal for different AQP2 COOH-terminal sites normalized for protein loading, as assessed by densitometry of the immunoblot. Values are means ± SE of 4 independent experiments. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

    doi: 10.1152/ajprenal.00308.2016

    Figure Lengend Snippet: Changes in AQP2 phosphorylation pattern following dDAVP treatment in the presence or absence of AMPK activator in mpkCCD c14 cells. Twenty-four hours posttransfection with wild-type AQP2 plasmid, cells were incubated in the presence or absence of AICAR (2 mM) for 4 h, and then dDAVP (10 nM) or vehicle was added for 30 min before cell harvesting. Immunoblot analysis of immunoprecipitated V5-AQP2 was performed using the corresponding rabbit polyclonal antibodies against phosphorylated AQP2 COOH-terminal sites: −S256, −S261, −S264, and −S269. To confirm successful immunoprecipitation, the membranes were reblotted using anti-AQP2 and anti-V5 antibodies coupled to HRP. A : representative set of immunoblots from these experiments using antibodies against AQP2 and its different COOH-terminal phosphorylation sites. B : quantification of AQP2 phosphorylation signal for different AQP2 COOH-terminal sites normalized for protein loading, as assessed by densitometry of the immunoblot. Values are means ± SE of 4 independent experiments. * P

    Article Snippet: The rabbit anti-AQP2 antibody was obtained from Alomone Labs (Jerusalem, Israel).

    Techniques: Plasmid Preparation, Incubation, Cell Harvesting, Immunoprecipitation, Western Blot

    Downregulation of AQP2 mRNA by LiCl is time and concentration dependent. IMCD cells were treated for different time points with LiCl (20 mM). The mRNA expression of AQP2-4, BGT1, and AR was analyzed by real-time PCR and the relative changes compared to untreated cells were calculated ( a ). In the same way, IMCD cells were treated for 24 h with 10 or 20 mM of LiCl and the relative changes in the gene expression of AQP2 and AR were compared to untreated cells ( b ). One-way ANOVA analysis with Tukey post-test, * indicates statistically significant differences to untreated cells ( p ≤ 0.05); n = 3.

    Journal: Cells

    Article Title: Lithium Chloride and GSK3 Inhibition Reduce Aquaporin-2 Expression in Primary Cultured Inner Medullary Collecting Duct Cells Due to Independent Mechanisms

    doi: 10.3390/cells9041060

    Figure Lengend Snippet: Downregulation of AQP2 mRNA by LiCl is time and concentration dependent. IMCD cells were treated for different time points with LiCl (20 mM). The mRNA expression of AQP2-4, BGT1, and AR was analyzed by real-time PCR and the relative changes compared to untreated cells were calculated ( a ). In the same way, IMCD cells were treated for 24 h with 10 or 20 mM of LiCl and the relative changes in the gene expression of AQP2 and AR were compared to untreated cells ( b ). One-way ANOVA analysis with Tukey post-test, * indicates statistically significant differences to untreated cells ( p ≤ 0.05); n = 3.

    Article Snippet: Antibodies raised against Aqp2-4 were obtained from Alomone Labs (Jerusalem, Israel) and antibodies directed against phosphorylated Aqp2 at position S256 were a generous gift from M. Knepper (National Institutes of Health, Bethesda, MD, USA).

    Techniques: Concentration Assay, Expressing, Real-time Polymerase Chain Reaction

    MG132 and bafilomycin induce downregulation of Aqp2 on the mRNA level. IMCD cells were treated for 1 or 24 h with bafilomycin (Baf. 100 nM) or MG132 (2 µM) and the mRNA expression of Aqp2-4, BGT-1, and AR was compared to untreated cells by real-time PCR. One-way ANOVA with tukey post-test was performed and * indicates statistically significant differences compared untreated cells ( p ≤ 0.05).

    Journal: Cells

    Article Title: Lithium Chloride and GSK3 Inhibition Reduce Aquaporin-2 Expression in Primary Cultured Inner Medullary Collecting Duct Cells Due to Independent Mechanisms

    doi: 10.3390/cells9041060

    Figure Lengend Snippet: MG132 and bafilomycin induce downregulation of Aqp2 on the mRNA level. IMCD cells were treated for 1 or 24 h with bafilomycin (Baf. 100 nM) or MG132 (2 µM) and the mRNA expression of Aqp2-4, BGT-1, and AR was compared to untreated cells by real-time PCR. One-way ANOVA with tukey post-test was performed and * indicates statistically significant differences compared untreated cells ( p ≤ 0.05).

    Article Snippet: Antibodies raised against Aqp2-4 were obtained from Alomone Labs (Jerusalem, Israel) and antibodies directed against phosphorylated Aqp2 at position S256 were a generous gift from M. Knepper (National Institutes of Health, Bethesda, MD, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Migration influences the localization of AQPs. Wound healing assays were performed with IMCD-cells cultivated at 600 mosmol/kg. A scratch was induced and after 4 h the cells were stained for AQP2–4 using specific antibodies. For the AQP2 staining the cells were incubated with dbcAMP to induce membrane localization. Alexa-488 labeled secondary antibodies were used for visualization. The nuclei were stained with DAPI. The upper row show cells away from the wound. The lower rows show cells at the leading edge. Scale bar = 20 µm, representative image chosen from 6 to 9 independent experiments.

    Journal: Scientific Reports

    Article Title: Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells

    doi: 10.1038/s41598-021-91369-y

    Figure Lengend Snippet: Migration influences the localization of AQPs. Wound healing assays were performed with IMCD-cells cultivated at 600 mosmol/kg. A scratch was induced and after 4 h the cells were stained for AQP2–4 using specific antibodies. For the AQP2 staining the cells were incubated with dbcAMP to induce membrane localization. Alexa-488 labeled secondary antibodies were used for visualization. The nuclei were stained with DAPI. The upper row show cells away from the wound. The lower rows show cells at the leading edge. Scale bar = 20 µm, representative image chosen from 6 to 9 independent experiments.

    Article Snippet: Antibodies raised against AQP2–4 were obtained from Alomone Labs (Jerusalem, Israel) .

    Techniques: Migration, Staining, Incubation, Labeling