na k atpase alpha 1  (Millipore)


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    Structured Review

    Millipore na k atpase alpha 1
    Representative immunofluorescence images of <t>Na,K-ATPase</t> <t>alpha-1,</t> -2, and −3 expression at the rat retina. Na,K-ATPase isoforms labeled with fluorescein in the left hand column images, vWF visualized with Texas red in the second column, nuclei are DAPI-labeled, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls when no primary antibody was used. Unique Na,K-ATPase alpha-1 -3 specific expression in the retinal cell layers is seen, similar to previous reports [ 23 , 44 ]. The arrow indicates alpha-2 expression in the capillaries at the retinal ganglion cell layer. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer are labeled for orientation on the alpha-3 DAPI image. Scale bar = 50 μm.
    Na K Atpase Alpha 1, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/na k atpase alpha 1/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    na k atpase alpha 1 - by Bioz Stars, 2020-09
    85/100 stars

    Images

    1) Product Images from "Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat"

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    Journal: Fluids and Barriers of the CNS

    doi: 10.1186/2045-8118-10-14

    Representative immunofluorescence images of Na,K-ATPase alpha-1, -2, and −3 expression at the rat retina. Na,K-ATPase isoforms labeled with fluorescein in the left hand column images, vWF visualized with Texas red in the second column, nuclei are DAPI-labeled, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls when no primary antibody was used. Unique Na,K-ATPase alpha-1 -3 specific expression in the retinal cell layers is seen, similar to previous reports [ 23 , 44 ]. The arrow indicates alpha-2 expression in the capillaries at the retinal ganglion cell layer. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer are labeled for orientation on the alpha-3 DAPI image. Scale bar = 50 μm.
    Figure Legend Snippet: Representative immunofluorescence images of Na,K-ATPase alpha-1, -2, and −3 expression at the rat retina. Na,K-ATPase isoforms labeled with fluorescein in the left hand column images, vWF visualized with Texas red in the second column, nuclei are DAPI-labeled, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls when no primary antibody was used. Unique Na,K-ATPase alpha-1 -3 specific expression in the retinal cell layers is seen, similar to previous reports [ 23 , 44 ]. The arrow indicates alpha-2 expression in the capillaries at the retinal ganglion cell layer. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer are labeled for orientation on the alpha-3 DAPI image. Scale bar = 50 μm.

    Techniques Used: Immunofluorescence, Expressing, Labeling

    Representative images of meningeal areas showing DAB-stained Na,K-ATPase alpha-1, alpha-3, CGRP, and control, as indicated in the sections. M: branch of middle meningeal artery; N: trigeminal nerve fiber; c: capillary. Scale bar = 50 μm.
    Figure Legend Snippet: Representative images of meningeal areas showing DAB-stained Na,K-ATPase alpha-1, alpha-3, CGRP, and control, as indicated in the sections. M: branch of middle meningeal artery; N: trigeminal nerve fiber; c: capillary. Scale bar = 50 μm.

    Techniques Used: Staining

    Specificity of Na,K-ATPase alpha-2 immunoreactivity at the choroid plexus. The CP was labeled with anti-alpha-2 ( A ) or with peptide pre-absorbed anti-alpha-2 ( B ). The subtraction image is in C . D : no primary control. Scale bar = 20 μm.
    Figure Legend Snippet: Specificity of Na,K-ATPase alpha-2 immunoreactivity at the choroid plexus. The CP was labeled with anti-alpha-2 ( A ) or with peptide pre-absorbed anti-alpha-2 ( B ). The subtraction image is in C . D : no primary control. Scale bar = 20 μm.

    Techniques Used: Labeling

    Fluorescence images of Na,K-ATPase alpha-3 and CGRP in meninges. In the top row, Na,K-ATPase alpha-3 is labeled with fluorescein in the left, CGRP is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the arrowhead triplet indicate a trigeminal nerve. The lower horizontal images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-3 is expressed at the nerve fiber, not on the capillary. Scale bar = 10 μm.
    Figure Legend Snippet: Fluorescence images of Na,K-ATPase alpha-3 and CGRP in meninges. In the top row, Na,K-ATPase alpha-3 is labeled with fluorescein in the left, CGRP is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the arrowhead triplet indicate a trigeminal nerve. The lower horizontal images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-3 is expressed at the nerve fiber, not on the capillary. Scale bar = 10 μm.

    Techniques Used: Fluorescence, Labeling, Staining

    Representative immunofluorescence images of a choroid plexus from the 3rd ventricle, sequentially acquired at 1 μm steps in a z-stack to reveal the cellular location of Na,K-ATPase alpha-1 and −2 isoforms. The same Na,K-ATPase expression patterns were found in the choroid plexuses in the lateral ventricles (data not shown). Texas red is used to visualize alpha-1 and fluorescein for alpha-2, and the nuclei are DAPI stained. Negative controls (without primary antibody; data not shown) had no membrane or cytoplasmic staining. Scale bar = 10 μm.
    Figure Legend Snippet: Representative immunofluorescence images of a choroid plexus from the 3rd ventricle, sequentially acquired at 1 μm steps in a z-stack to reveal the cellular location of Na,K-ATPase alpha-1 and −2 isoforms. The same Na,K-ATPase expression patterns were found in the choroid plexuses in the lateral ventricles (data not shown). Texas red is used to visualize alpha-1 and fluorescein for alpha-2, and the nuclei are DAPI stained. Negative controls (without primary antibody; data not shown) had no membrane or cytoplasmic staining. Scale bar = 10 μm.

    Techniques Used: Immunofluorescence, Expressing, Staining

    Representative immunofluorescence images of the blood-aqueous barrier at the rat ciliary body with Na,K-ATPase alpha-1, -2, and −3 isoform expression. Na,K-ATPase isoforms labeled with fluorescein in left hand column images, vWF visualized with Texas red in second column, nuclei are DAPI stained, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls with no primary antibody. Merged images show alpha-1 expressed on the PE, alpha-2 on the NPE, and alpha-2 expressed on ciliary body capillaries. The arrowhead points to the PE on the alpha-1 merged image, and the arrow points to the NPE on the alpha-2 merged image. Scale bar = 20 μm.
    Figure Legend Snippet: Representative immunofluorescence images of the blood-aqueous barrier at the rat ciliary body with Na,K-ATPase alpha-1, -2, and −3 isoform expression. Na,K-ATPase isoforms labeled with fluorescein in left hand column images, vWF visualized with Texas red in second column, nuclei are DAPI stained, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls with no primary antibody. Merged images show alpha-1 expressed on the PE, alpha-2 on the NPE, and alpha-2 expressed on ciliary body capillaries. The arrowhead points to the PE on the alpha-1 merged image, and the arrow points to the NPE on the alpha-2 merged image. Scale bar = 20 μm.

    Techniques Used: Immunofluorescence, Expressing, Labeling, Staining

    A is a tiled DAB image of Na,K-ATPase alpha-2 immunostaining at the rat meninges. The field of view is half of the meninges, extending from the transverse sinus (TS) to the superior sagittal sinus (SSS) centrally and the middle meningeal artery (MMA) laterally, with the trigeminal nerve fibers (TN) crossing the dura. Scale bar 500 μm. B : Enlarged region of meninges, magnified from the rectangle area in Figure 9 A, illustrating branches of the MMA, a TN, and a capillary (DCap). Scale bar = 50 μm. For control, see Figure 10 .
    Figure Legend Snippet: A is a tiled DAB image of Na,K-ATPase alpha-2 immunostaining at the rat meninges. The field of view is half of the meninges, extending from the transverse sinus (TS) to the superior sagittal sinus (SSS) centrally and the middle meningeal artery (MMA) laterally, with the trigeminal nerve fibers (TN) crossing the dura. Scale bar 500 μm. B : Enlarged region of meninges, magnified from the rectangle area in Figure 9 A, illustrating branches of the MMA, a TN, and a capillary (DCap). Scale bar = 50 μm. For control, see Figure 10 .

    Techniques Used: Immunostaining

    Fluorescence images of CGRP and Na,K-ATPase alpha-2 in meninges. In the top row, CGRP is labeled with fluorescein on the left, Na,K-ATPase alpha-2 is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the smaller arrowhead indicates a trigeminal nerve. The lower row images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-2 is expressed at the capillary and nerve fiber. Scale bar = 10 μm.
    Figure Legend Snippet: Fluorescence images of CGRP and Na,K-ATPase alpha-2 in meninges. In the top row, CGRP is labeled with fluorescein on the left, Na,K-ATPase alpha-2 is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the smaller arrowhead indicates a trigeminal nerve. The lower row images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-2 is expressed at the capillary and nerve fiber. Scale bar = 10 μm.

    Techniques Used: Fluorescence, Labeling, Staining

    Specificity of Na,K-ATPase alpha-2 immunoreactivity at the retina. The retina was stained with anti-alpha-2 ( A ) or with pre-absorbed anti-alpha-2 ( B ). Subtraction image is shown in C . D : no primary negative control. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer; asterisk: ganglion cell layer; small arrow: RPE. Scale bar = 20 μm.
    Figure Legend Snippet: Specificity of Na,K-ATPase alpha-2 immunoreactivity at the retina. The retina was stained with anti-alpha-2 ( A ) or with pre-absorbed anti-alpha-2 ( B ). Subtraction image is shown in C . D : no primary negative control. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer; asterisk: ganglion cell layer; small arrow: RPE. Scale bar = 20 μm.

    Techniques Used: Staining, Negative Control

    Related Articles

    Immunohistochemistry:

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat
    Article Snippet: .. Immunohistochemistry Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). ..

    Labeling:

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat
    Article Snippet: .. Immunohistochemistry Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). ..

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    Millipore anti na k atpase alpha antibody
    Effect of short-term renal interstitial coinfusion of βMCD on D 1 -like receptor-induced internalization of <t>Na-K-ATPase.</t> At the end of the acute study shown in , kidneys from the rats were removed and cortical tissue lysates were made. Adaptor
    Anti Na K Atpase Alpha Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti na k atpase alpha antibody/product/Millipore
    Average 96 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
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    Millipore anti na k atpase α 1
    ARL-13/Arl13b associates with ciliary membranes via palmitoyl anchors. (A) N-terminal Pal motif ARL-13 variants (delPal and rPal) fail to localize to amphid (head) and phasmid (tail) cilia (c) and diffusely mislocalize in cell bodies (cb) and dendrites (d). C-terminal deletion ARL-13(del203–370) is also mislocalized, but membrane associations are maintained (arrows). Insets show high magnification images of PHA/B cilia. (B) HsArl13b ciliary localization requires an N-terminal Pal motif. Ciliated RPE1 cells transfected with GFP-tagged Arl13b(WT) or a C8S/C9S variant and costained for ciliary axonemes using acetylated α-tubulin antibody are shown. Merged images show that Arl13b(C8S/C9S) is highly diffuse and nonmembrane associated, with weak signals in cilia. The boxed regions are shown at high magnification in the insets. Dashed lines outline the cell. (C) Loss of Pal motif shifts HsArl13b to cytosolic fractions. A subcellular fractionation scheme for Flag-tagged Arl13b (WT and variants) transiently expressed in 293T cells is shown. Supernatants (Sup.) and pellets (Pell) were probed for Arl13b::Flag using Western blotting and an anti-Flag antibody. Calpain and Na + /K + <t>ATPase</t> proteins mark cytosol and membrane fractions, respectively. Arl13b(WT) and Arl13b(1–357) pellet with Na + /K + ATPase membrane proteins after 20,000 g spins, whereas Arl13b(C8S/C9S) remains in calpain-enriched cytosolic supernatants even after 200,000 g spins. (D) Arl13b is palmitoylated in 293T cells. 293T cells transiently transfected with GFP-tagged Arl13b(WT) or Arl13b(C8S/C9S) were metabolically labeled with [ 3 H]palmitate, and proteins were separated by SDS-PAGE. The top panel shows a fluorograph; the bottom panel shows a Western blot with anti-GFP antibody. The arrow indicates palmitoylated Arl13b-GFP, and the asterisk indicates an unknown endogenous palmitoylated protein. Bars: (A) 5 µm; (B) 10 µm.
    Anti Na K Atpase α 1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti na k atpase α 1/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti na k atpase α 1 - by Bioz Stars, 2020-09
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    Image Search Results


    Effect of short-term renal interstitial coinfusion of βMCD on D 1 -like receptor-induced internalization of Na-K-ATPase. At the end of the acute study shown in , kidneys from the rats were removed and cortical tissue lysates were made. Adaptor

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Inhibition of renal caveolin-1 reduces natriuresis and produces hypertension in sodium-loaded rats

    doi: 10.1152/ajprenal.00380.2010

    Figure Lengend Snippet: Effect of short-term renal interstitial coinfusion of βMCD on D 1 -like receptor-induced internalization of Na-K-ATPase. At the end of the acute study shown in , kidneys from the rats were removed and cortical tissue lysates were made. Adaptor

    Article Snippet: The eluate was immuno-dot-blotted for Na-K-ATPase using a 1:1,000 dilution of anti-Na-K-ATPase alpha antibody (Millipore clone C464.6).

    Techniques:

    Immunoblot results showing MC2-R and MC3-R expression in aorta-derived mesenchymal cell lines. ( A ) A representative blot showing MC2-R detection in crude membrane fractions of mouse aorta cells mAo) untreated (−) or treated (+) with 10 nM Dex; the clonal mouse aorta-derived cell line (C8) untreated (−) or treated (+) with 10 nM Dex; and rat bone marrow derived MSC induced to form adipocytes (Adip) as a control (Adip con). ( B ) A representative blot showing detection of MC3-R in crude membrane fractions of mAo cells untreated (−) or treated (+) with 10 nM Dex and C8 cells untreated (−) or treated (+) with 10 nM Dex. Adip were used as a negative control (neg con). ( C ) A representative blot showing the absence of MC5-R in crude membrane fractions of mAo cells untreated (lane 1) or treated with 10 nM Dex (lane 2) and C8 cells untreated untreated (−) or treated (+) with 10 nM Dex Adip were used as a positive control (pos con). SDS-PAGE gels were loaded with 50 µg protein crude membrane fraction per lane. Detection of Na + /K + ATPase was used as a control for gel loading. ( D ) Densitometric analysis of MC2-R and MC3-R immunoblots. Data are presented as mean % expression ± SD of untreated (− Dex) cultures, n = 3–4.

    Journal: Molecular and cellular endocrinology

    Article Title: Functional melanocortin-2 receptors are expressed by mouse aorta-derived mesenchymal progenitor cells

    doi: 10.1016/j.mce.2012.01.019

    Figure Lengend Snippet: Immunoblot results showing MC2-R and MC3-R expression in aorta-derived mesenchymal cell lines. ( A ) A representative blot showing MC2-R detection in crude membrane fractions of mouse aorta cells mAo) untreated (−) or treated (+) with 10 nM Dex; the clonal mouse aorta-derived cell line (C8) untreated (−) or treated (+) with 10 nM Dex; and rat bone marrow derived MSC induced to form adipocytes (Adip) as a control (Adip con). ( B ) A representative blot showing detection of MC3-R in crude membrane fractions of mAo cells untreated (−) or treated (+) with 10 nM Dex and C8 cells untreated (−) or treated (+) with 10 nM Dex. Adip were used as a negative control (neg con). ( C ) A representative blot showing the absence of MC5-R in crude membrane fractions of mAo cells untreated (lane 1) or treated with 10 nM Dex (lane 2) and C8 cells untreated untreated (−) or treated (+) with 10 nM Dex Adip were used as a positive control (pos con). SDS-PAGE gels were loaded with 50 µg protein crude membrane fraction per lane. Detection of Na + /K + ATPase was used as a control for gel loading. ( D ) Densitometric analysis of MC2-R and MC3-R immunoblots. Data are presented as mean % expression ± SD of untreated (− Dex) cultures, n = 3–4.

    Article Snippet: The monoclonal Na+/K+ ATPase α-1 antibody (clone C464.6) was from Millipore (Temeculae, CA) and the β-Actin monoclonal antibody from SIGMA-Aldrich (USA).

    Techniques: Expressing, Derivative Assay, Negative Control, Positive Control, SDS Page, Western Blot

    Inhibition of the Gαi3 protein, localized in AQP2 vesicles, prevents the Forskolin-induced increase in water permeability. ( A ) Expression of AQP2, Gαi3, Vamp2 and Na + /K + -ATPase in MCD4 cell homogenate and in AQP2 vesicles purified from MCD4. 30 μg of protein from total homogenate (H) or AQP2 vesicles (V) were blotted and probed with indicated antibodies. AQP2 is found highly enriched in the vesicle fraction along with the vesicle marker VAMP2, with low contamination by plasma membranes tested using the marker Na + /K + -ATPaseα1. Gαi3 appears to be expressed both in total homogenate and in vesicles fraction. Western blot is representative of 3 experiments. ( B ) Immunofluorescence localization of AQP2 and Gαi3 in polarized MCD4 cells. Representative confocal images showing substantial colocalization between AQP2 (green) and Gαi3 (red) evident upon overlay of individual fluorescence channels. ( C ) Effect of PTX treatment on osmotic water permeability under FK action in MCD4 cells. Cell monolayers were treated with Forskolin (100 μM) and/or PTX (2 μg/mL) as described in Experimental Procedures. The time course of fluorescence changes in calcein-loaded cells indicates that in cells treated with FK (100 µM) a significant increase in osmotic water permeability reported as Ki (193.3 ± 11.22 s −1 , **** p

    Journal: Cells

    Article Title: Gi Protein Modulation of the Potassium Channel TASK-2 Mediates Vesicle Osmotic Swelling to Facilitate the Fusion of Aquaporin-2 Water Channel Containing Vesicles

    doi: 10.3390/cells7120276

    Figure Lengend Snippet: Inhibition of the Gαi3 protein, localized in AQP2 vesicles, prevents the Forskolin-induced increase in water permeability. ( A ) Expression of AQP2, Gαi3, Vamp2 and Na + /K + -ATPase in MCD4 cell homogenate and in AQP2 vesicles purified from MCD4. 30 μg of protein from total homogenate (H) or AQP2 vesicles (V) were blotted and probed with indicated antibodies. AQP2 is found highly enriched in the vesicle fraction along with the vesicle marker VAMP2, with low contamination by plasma membranes tested using the marker Na + /K + -ATPaseα1. Gαi3 appears to be expressed both in total homogenate and in vesicles fraction. Western blot is representative of 3 experiments. ( B ) Immunofluorescence localization of AQP2 and Gαi3 in polarized MCD4 cells. Representative confocal images showing substantial colocalization between AQP2 (green) and Gαi3 (red) evident upon overlay of individual fluorescence channels. ( C ) Effect of PTX treatment on osmotic water permeability under FK action in MCD4 cells. Cell monolayers were treated with Forskolin (100 μM) and/or PTX (2 μg/mL) as described in Experimental Procedures. The time course of fluorescence changes in calcein-loaded cells indicates that in cells treated with FK (100 µM) a significant increase in osmotic water permeability reported as Ki (193.3 ± 11.22 s −1 , **** p

    Article Snippet: Anti-Na+ /K+ -ATPase subunit alpha 1 (clone C464.6) was from Millipore (Milano, Italy).

    Techniques: Inhibition, Permeability, Expressing, Purification, Marker, Western Blot, Immunofluorescence, Fluorescence

    Heterotrimeric G protein Gαi3 is associated with AQP2 vesicles isolated from rat kidney medulla. ( A ) Expression of AQP2, Gαi3, Vamp2 and Na + /K + -ATPase in rat kidney medulla homogenate and AQP2 vesicles isolated from rat kidney medulla. 30 μg of protein from total rat homogenate (H) or purified AQP2 vesicles from rat inner kidney medulla (V) were blotted and probed with indicated antibodies. AQP2 is found highly enriched in the vesicle fraction along with the vesicle marker VAMP2, with low contamination by plasma membranes tested using the marker Na + /K + -ATPaseα1. Gαi3 appears to be expressed both in total homogenate and in vesicles fraction. Western blot is representative of 3 experiments. ( B ) Localization of AQP2 and Gαi3 in rat kidney. Representative confocal images showing substantial colocalization between AQP2 (green) and Gαi3 (red) evident upon overlay of individual fluorescence channels. The two proteins display a predominant punctuate intracellular localization. ( C ) Subcellular distribution of Gαi3 and AQP2 under basal condition or stimulated with dDAVP (1µM for 45 min) in rat kidney. Rat kidney slices were stimulated with dDAVP or left under basal conditions, homogenized and subjected to differential centrifugation to obtain plasma membrane, intracellular vesicles and cytosol. Protein samples were blotted and probed with specific antibodies against AQP2 and Gαi3. ( D ) Statistical analysis of Gαi3 and AQP2 expression normalized to total protein in membrane (M), vesicle fraction (V) and total homogenate (H) is reported. Values are reported as means ± S.E.Ms; *** p

    Journal: Cells

    Article Title: Gi Protein Modulation of the Potassium Channel TASK-2 Mediates Vesicle Osmotic Swelling to Facilitate the Fusion of Aquaporin-2 Water Channel Containing Vesicles

    doi: 10.3390/cells7120276

    Figure Lengend Snippet: Heterotrimeric G protein Gαi3 is associated with AQP2 vesicles isolated from rat kidney medulla. ( A ) Expression of AQP2, Gαi3, Vamp2 and Na + /K + -ATPase in rat kidney medulla homogenate and AQP2 vesicles isolated from rat kidney medulla. 30 μg of protein from total rat homogenate (H) or purified AQP2 vesicles from rat inner kidney medulla (V) were blotted and probed with indicated antibodies. AQP2 is found highly enriched in the vesicle fraction along with the vesicle marker VAMP2, with low contamination by plasma membranes tested using the marker Na + /K + -ATPaseα1. Gαi3 appears to be expressed both in total homogenate and in vesicles fraction. Western blot is representative of 3 experiments. ( B ) Localization of AQP2 and Gαi3 in rat kidney. Representative confocal images showing substantial colocalization between AQP2 (green) and Gαi3 (red) evident upon overlay of individual fluorescence channels. The two proteins display a predominant punctuate intracellular localization. ( C ) Subcellular distribution of Gαi3 and AQP2 under basal condition or stimulated with dDAVP (1µM for 45 min) in rat kidney. Rat kidney slices were stimulated with dDAVP or left under basal conditions, homogenized and subjected to differential centrifugation to obtain plasma membrane, intracellular vesicles and cytosol. Protein samples were blotted and probed with specific antibodies against AQP2 and Gαi3. ( D ) Statistical analysis of Gαi3 and AQP2 expression normalized to total protein in membrane (M), vesicle fraction (V) and total homogenate (H) is reported. Values are reported as means ± S.E.Ms; *** p

    Article Snippet: Anti-Na+ /K+ -ATPase subunit alpha 1 (clone C464.6) was from Millipore (Milano, Italy).

    Techniques: Isolation, Expressing, Purification, Marker, Western Blot, Fluorescence, Centrifugation, Mass Spectrometry

    ARL-13/Arl13b associates with ciliary membranes via palmitoyl anchors. (A) N-terminal Pal motif ARL-13 variants (delPal and rPal) fail to localize to amphid (head) and phasmid (tail) cilia (c) and diffusely mislocalize in cell bodies (cb) and dendrites (d). C-terminal deletion ARL-13(del203–370) is also mislocalized, but membrane associations are maintained (arrows). Insets show high magnification images of PHA/B cilia. (B) HsArl13b ciliary localization requires an N-terminal Pal motif. Ciliated RPE1 cells transfected with GFP-tagged Arl13b(WT) or a C8S/C9S variant and costained for ciliary axonemes using acetylated α-tubulin antibody are shown. Merged images show that Arl13b(C8S/C9S) is highly diffuse and nonmembrane associated, with weak signals in cilia. The boxed regions are shown at high magnification in the insets. Dashed lines outline the cell. (C) Loss of Pal motif shifts HsArl13b to cytosolic fractions. A subcellular fractionation scheme for Flag-tagged Arl13b (WT and variants) transiently expressed in 293T cells is shown. Supernatants (Sup.) and pellets (Pell) were probed for Arl13b::Flag using Western blotting and an anti-Flag antibody. Calpain and Na + /K + ATPase proteins mark cytosol and membrane fractions, respectively. Arl13b(WT) and Arl13b(1–357) pellet with Na + /K + ATPase membrane proteins after 20,000 g spins, whereas Arl13b(C8S/C9S) remains in calpain-enriched cytosolic supernatants even after 200,000 g spins. (D) Arl13b is palmitoylated in 293T cells. 293T cells transiently transfected with GFP-tagged Arl13b(WT) or Arl13b(C8S/C9S) were metabolically labeled with [ 3 H]palmitate, and proteins were separated by SDS-PAGE. The top panel shows a fluorograph; the bottom panel shows a Western blot with anti-GFP antibody. The arrow indicates palmitoylated Arl13b-GFP, and the asterisk indicates an unknown endogenous palmitoylated protein. Bars: (A) 5 µm; (B) 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans

    doi: 10.1083/jcb.200908133

    Figure Lengend Snippet: ARL-13/Arl13b associates with ciliary membranes via palmitoyl anchors. (A) N-terminal Pal motif ARL-13 variants (delPal and rPal) fail to localize to amphid (head) and phasmid (tail) cilia (c) and diffusely mislocalize in cell bodies (cb) and dendrites (d). C-terminal deletion ARL-13(del203–370) is also mislocalized, but membrane associations are maintained (arrows). Insets show high magnification images of PHA/B cilia. (B) HsArl13b ciliary localization requires an N-terminal Pal motif. Ciliated RPE1 cells transfected with GFP-tagged Arl13b(WT) or a C8S/C9S variant and costained for ciliary axonemes using acetylated α-tubulin antibody are shown. Merged images show that Arl13b(C8S/C9S) is highly diffuse and nonmembrane associated, with weak signals in cilia. The boxed regions are shown at high magnification in the insets. Dashed lines outline the cell. (C) Loss of Pal motif shifts HsArl13b to cytosolic fractions. A subcellular fractionation scheme for Flag-tagged Arl13b (WT and variants) transiently expressed in 293T cells is shown. Supernatants (Sup.) and pellets (Pell) were probed for Arl13b::Flag using Western blotting and an anti-Flag antibody. Calpain and Na + /K + ATPase proteins mark cytosol and membrane fractions, respectively. Arl13b(WT) and Arl13b(1–357) pellet with Na + /K + ATPase membrane proteins after 20,000 g spins, whereas Arl13b(C8S/C9S) remains in calpain-enriched cytosolic supernatants even after 200,000 g spins. (D) Arl13b is palmitoylated in 293T cells. 293T cells transiently transfected with GFP-tagged Arl13b(WT) or Arl13b(C8S/C9S) were metabolically labeled with [ 3 H]palmitate, and proteins were separated by SDS-PAGE. The top panel shows a fluorograph; the bottom panel shows a Western blot with anti-GFP antibody. The arrow indicates palmitoylated Arl13b-GFP, and the asterisk indicates an unknown endogenous palmitoylated protein. Bars: (A) 5 µm; (B) 10 µm.

    Article Snippet: The cell suspension was homogenized by 10 strokes in a chilled cell homogenizer with a tungsten-carbide ball (clearance of 10 µm; Isobiotec) and then subjected to standard subcellular fractionation by centrifugation , followed by immunoblotting with anti-Flag M2, anti–µ-calpain (#MA3-940; Thermo Fisher Scientific), and anti-Na+ /K+ ATPase α-1 (Millipore) antibodies.

    Techniques: Transfection, Variant Assay, Fractionation, Western Blot, Metabolic Labelling, Labeling, SDS Page