Structured Review

Thermo Fisher v atpase
<t>V-ATPase</t> regulates ciliogenesis in multiple organs in zebrafish. (A) Embryonic expression of Vod1 at day 1 as detected by in situ hybridization. It is widely distributed. (B) Knockdown of Vod1 by morpholino impairs the pigmentation in the trunk but not the retina. Similar defect is observed in V1D morphants (data not shown). (C) Morpholino knockdown of Vod1 causes heart-looping defect similar to that in SNX10a morphants. Furthermore, Vod1 and <t>SNX10</t> function synergistically in the left-right patterning of heart. 10a-SP at 1.0 ng/embryo or Vod1-AUG at 0.75 ng/embryo alone cannot effectively induce heart-looping defect, however, the combination of them is sufficient to disrupt the heart-looping process. (D) Inhibition of V-ATPase induces the formation of pronephric cyst in 65% of the treated embryos ( n = 46). Picture shows a cross-section of Vod1 morphant at the position of the cyst (arrows). NC: notochord. (E - G) V-ATPase is involved in ciliogenesis in multiple organs in zebrafish. Cilia in KV ( E , 10-somite stage), pronephric duct ( F , 27 hpf) or hair cells of lateral line neuromasts (red staining in G , day 2) are visualized by whole-mount fluorescence immunostaining. The nuclei are counter-stained with DAPI (blue). Ciliogenesis in the KV of V-ATPase morphants are severely inhibited. The cilia in pronephric duct are disorganized and reduced in number when V-ATPase is blocked. The neuromasts are relatively normal in the morphants (blue staining in G ), but their hair cells fail to generate cilia.
V Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo"

Article Title: A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo

Journal: Cell Research

doi: 10.1038/cr.2011.134

V-ATPase regulates ciliogenesis in multiple organs in zebrafish. (A) Embryonic expression of Vod1 at day 1 as detected by in situ hybridization. It is widely distributed. (B) Knockdown of Vod1 by morpholino impairs the pigmentation in the trunk but not the retina. Similar defect is observed in V1D morphants (data not shown). (C) Morpholino knockdown of Vod1 causes heart-looping defect similar to that in SNX10a morphants. Furthermore, Vod1 and SNX10 function synergistically in the left-right patterning of heart. 10a-SP at 1.0 ng/embryo or Vod1-AUG at 0.75 ng/embryo alone cannot effectively induce heart-looping defect, however, the combination of them is sufficient to disrupt the heart-looping process. (D) Inhibition of V-ATPase induces the formation of pronephric cyst in 65% of the treated embryos ( n = 46). Picture shows a cross-section of Vod1 morphant at the position of the cyst (arrows). NC: notochord. (E - G) V-ATPase is involved in ciliogenesis in multiple organs in zebrafish. Cilia in KV ( E , 10-somite stage), pronephric duct ( F , 27 hpf) or hair cells of lateral line neuromasts (red staining in G , day 2) are visualized by whole-mount fluorescence immunostaining. The nuclei are counter-stained with DAPI (blue). Ciliogenesis in the KV of V-ATPase morphants are severely inhibited. The cilia in pronephric duct are disorganized and reduced in number when V-ATPase is blocked. The neuromasts are relatively normal in the morphants (blue staining in G ), but their hair cells fail to generate cilia.
Figure Legend Snippet: V-ATPase regulates ciliogenesis in multiple organs in zebrafish. (A) Embryonic expression of Vod1 at day 1 as detected by in situ hybridization. It is widely distributed. (B) Knockdown of Vod1 by morpholino impairs the pigmentation in the trunk but not the retina. Similar defect is observed in V1D morphants (data not shown). (C) Morpholino knockdown of Vod1 causes heart-looping defect similar to that in SNX10a morphants. Furthermore, Vod1 and SNX10 function synergistically in the left-right patterning of heart. 10a-SP at 1.0 ng/embryo or Vod1-AUG at 0.75 ng/embryo alone cannot effectively induce heart-looping defect, however, the combination of them is sufficient to disrupt the heart-looping process. (D) Inhibition of V-ATPase induces the formation of pronephric cyst in 65% of the treated embryos ( n = 46). Picture shows a cross-section of Vod1 morphant at the position of the cyst (arrows). NC: notochord. (E - G) V-ATPase is involved in ciliogenesis in multiple organs in zebrafish. Cilia in KV ( E , 10-somite stage), pronephric duct ( F , 27 hpf) or hair cells of lateral line neuromasts (red staining in G , day 2) are visualized by whole-mount fluorescence immunostaining. The nuclei are counter-stained with DAPI (blue). Ciliogenesis in the KV of V-ATPase morphants are severely inhibited. The cilia in pronephric duct are disorganized and reduced in number when V-ATPase is blocked. The neuromasts are relatively normal in the morphants (blue staining in G ), but their hair cells fail to generate cilia.

Techniques Used: Expressing, In Situ Hybridization, Inhibition, Staining, Fluorescence, Immunostaining

V-ATPase binds to SNX10 and mediates SNX10-induced vacuole formation. (A) V-ATPase is required for SNX10 to induce vacuoles in Hela cells. Pretreatment of cells with siRNA to the Vod1subunit of V-ATPase inhibits the formation of SNX10-GFP(10G)-induced vacuoles. Treatment of cells with the V-ATPase inhibitor bafilomycin A1 (baf) also inhibits the SNX10-GFP-induced vacuoles. (B) SNX10 co-localizes with V-ATPase. SNX10-GFP is co-transfected into RCC10/VHL cells with the HA-tagged Vod1 or V1D subunit of V-ATPase. Cells are fixed 24 h after transfection and immunostained with the mouse anti-HA antibody (red). Either subunit of the V-ATPase complex co-localizes with a fraction of SNX10-GFP. (C) SNX10 co-immunoprecipitates with the V1D subunit of V-ATPase. Left panel: HA-tagged SNX10 is co-transfected into 293T cells with the Flag-tagged GFP or Flag-GFP-tagged V1D. Cells are harvested 36 h after transfection and immunoprecipitation was performed with the resin-conjugated anti-Flag antibody. The levels of HA-SNX10 in cell lysates and immunoprecipitated complexes are detected by western blot using the mouse anti-HA IgG. V1D but not the Flag-GFP can pull down the HA-SNX10. Middle panel: Flag-GFP-tagged full-length SNX10 (10G) or the PX domain of SNX10 (PX, a.a. 8-125), but not the C-terminal domain of SNX10 (CD, a.a. 126-202), co-immunoprecipitates with HA-V1D. SNX16 (16G), another PX domain protein, does not pull down V1D. Right panel: HA-tagged V1A (A) and V1C (C) subunit of V-ATPase do not bind to SNX10. When they are co-expressed with V1D (D), SNX10 only pulls-down the V1D.
Figure Legend Snippet: V-ATPase binds to SNX10 and mediates SNX10-induced vacuole formation. (A) V-ATPase is required for SNX10 to induce vacuoles in Hela cells. Pretreatment of cells with siRNA to the Vod1subunit of V-ATPase inhibits the formation of SNX10-GFP(10G)-induced vacuoles. Treatment of cells with the V-ATPase inhibitor bafilomycin A1 (baf) also inhibits the SNX10-GFP-induced vacuoles. (B) SNX10 co-localizes with V-ATPase. SNX10-GFP is co-transfected into RCC10/VHL cells with the HA-tagged Vod1 or V1D subunit of V-ATPase. Cells are fixed 24 h after transfection and immunostained with the mouse anti-HA antibody (red). Either subunit of the V-ATPase complex co-localizes with a fraction of SNX10-GFP. (C) SNX10 co-immunoprecipitates with the V1D subunit of V-ATPase. Left panel: HA-tagged SNX10 is co-transfected into 293T cells with the Flag-tagged GFP or Flag-GFP-tagged V1D. Cells are harvested 36 h after transfection and immunoprecipitation was performed with the resin-conjugated anti-Flag antibody. The levels of HA-SNX10 in cell lysates and immunoprecipitated complexes are detected by western blot using the mouse anti-HA IgG. V1D but not the Flag-GFP can pull down the HA-SNX10. Middle panel: Flag-GFP-tagged full-length SNX10 (10G) or the PX domain of SNX10 (PX, a.a. 8-125), but not the C-terminal domain of SNX10 (CD, a.a. 126-202), co-immunoprecipitates with HA-V1D. SNX16 (16G), another PX domain protein, does not pull down V1D. Right panel: HA-tagged V1A (A) and V1C (C) subunit of V-ATPase do not bind to SNX10. When they are co-expressed with V1D (D), SNX10 only pulls-down the V1D.

Techniques Used: Transfection, Immunoprecipitation, Western Blot

SNX10 regulates the intracellular trafficking of V-ATPase and Rab8a. (A) SNX10 regulates the centrosomal localization of V-ATPase. RCC10/VHL cells are first treated with siRNAs to SNX10 for 24 h, plasmids for Vod1-GFP or V1D-GFP are then transfected into these cells. Cells are fixed and immunostained for Pericentrin after 24 h. In control siRNA-treated cells, a fraction of Vod1-GFP co-localizes with Pericentrin (red). In SNX10 siRNAs-treated cells, the centrosomal localization of Vod1-GFP is reduced. Vod1-GFP at other subcellular locations appears not affected. (B) Statistical analysis of A . (C) Western blot analysis for Vod1-GFP protein level with an anti-GFP antibody. The expression level of Vod1-GFP is not affected by the indicated siRNAs. Tubulin is the loading control. (D) Inhibition of SNX10 or V-ATPase does not affect the co-localization of Pericentrin and γ-tubulin. (E) Subcellular localization of SNX10 and Rab8a. Rab8a-GFP is enriched at the pericentriolar region and SNX10 vesicles are observed around this region. (F) SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a. The Rab8a-GFP stable cell line is treated with the indicated siRNAs for 24 h then serum starved for another 24 h. Cells are then fixed for immunostainig. In the control siRNA-treated cells, 43% of the cilia is Rab8a-GFP positive. When SNX10 or V-ATPase is knocked down, in a subset of cells that form cilia, the ratio of Rab8a positive cilia is reduced. (G) Statistical analysis of F . The ratio of Rab8a positive cilia is reduced to less than 21% in all cases when SNX10 or V-ATPase is inhibited ( P
Figure Legend Snippet: SNX10 regulates the intracellular trafficking of V-ATPase and Rab8a. (A) SNX10 regulates the centrosomal localization of V-ATPase. RCC10/VHL cells are first treated with siRNAs to SNX10 for 24 h, plasmids for Vod1-GFP or V1D-GFP are then transfected into these cells. Cells are fixed and immunostained for Pericentrin after 24 h. In control siRNA-treated cells, a fraction of Vod1-GFP co-localizes with Pericentrin (red). In SNX10 siRNAs-treated cells, the centrosomal localization of Vod1-GFP is reduced. Vod1-GFP at other subcellular locations appears not affected. (B) Statistical analysis of A . (C) Western blot analysis for Vod1-GFP protein level with an anti-GFP antibody. The expression level of Vod1-GFP is not affected by the indicated siRNAs. Tubulin is the loading control. (D) Inhibition of SNX10 or V-ATPase does not affect the co-localization of Pericentrin and γ-tubulin. (E) Subcellular localization of SNX10 and Rab8a. Rab8a-GFP is enriched at the pericentriolar region and SNX10 vesicles are observed around this region. (F) SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a. The Rab8a-GFP stable cell line is treated with the indicated siRNAs for 24 h then serum starved for another 24 h. Cells are then fixed for immunostainig. In the control siRNA-treated cells, 43% of the cilia is Rab8a-GFP positive. When SNX10 or V-ATPase is knocked down, in a subset of cells that form cilia, the ratio of Rab8a positive cilia is reduced. (G) Statistical analysis of F . The ratio of Rab8a positive cilia is reduced to less than 21% in all cases when SNX10 or V-ATPase is inhibited ( P

Techniques Used: Transfection, Western Blot, Expressing, Inhibition, Stable Transfection

2) Product Images from "Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1"

Article Title: Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.202115499

Stability of Pma1 in lcb1 - 100 cells. ( A ) Western blot of HA-Pma1. LCB1 + (F1105) , lcb1 - 100 (RHY3804), and lcb1 - 100 pep4 (WQY7) cells bearing GAL-HA-PMA1 (pND542) were grown in medium with raffinose (0), shifted to galactose for 4 h at 30°C (on), and incubated for an additional 4 h after adding 3% glucose (off). Western blot was quantitated with NIH IMAGE and plotted as a percent of HA-Pma1 synthesized. Half-life of Pma1 is decreased in lcb1 cells; Pma1 degradation is dependent on Pep4. ( B ) Indirect immunofluorescence localization of Pma1. lcb1 - 100 (RH3804) growing at 30°C were fixed, permeabilized, stained with anti-Pma1 and V-ATPase, followed by Cy2- and Cy3-conjugated secondary antibodies.
Figure Legend Snippet: Stability of Pma1 in lcb1 - 100 cells. ( A ) Western blot of HA-Pma1. LCB1 + (F1105) , lcb1 - 100 (RHY3804), and lcb1 - 100 pep4 (WQY7) cells bearing GAL-HA-PMA1 (pND542) were grown in medium with raffinose (0), shifted to galactose for 4 h at 30°C (on), and incubated for an additional 4 h after adding 3% glucose (off). Western blot was quantitated with NIH IMAGE and plotted as a percent of HA-Pma1 synthesized. Half-life of Pma1 is decreased in lcb1 cells; Pma1 degradation is dependent on Pep4. ( B ) Indirect immunofluorescence localization of Pma1. lcb1 - 100 (RH3804) growing at 30°C were fixed, permeabilized, stained with anti-Pma1 and V-ATPase, followed by Cy2- and Cy3-conjugated secondary antibodies.

Techniques Used: Western Blot, Incubation, Synthesized, Immunofluorescence, Staining

3) Product Images from "The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling"

Article Title: The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.347

a2V expression during normal mammary development. Inguinal mammary glands from puberty, virginity, pregnancy, lactation and involution stages were harvested and analyzed for gene expression and cellular distribution of a2V. Total RNA extracted from mammary glands was subjected to quantitative real-time-PCR (qRT-PCR) analysis. ( a ) Gene expression of V-ATPase ‘a2' subunit isoform during various stages of gland development compared with puberty. ( b ) Relative gene expression of V-ATPase a1, a2 and a3 subunit isoforms compared with each other at every stage. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as endogenous housekeeping control. Data represent mean±S.E., n =6. * P ≤0.05, ** P ≤0.01, *** P ≤0.001 and **** P ≤0.0001. ( c ) Representative z-stack images from a confocal laser-scanning microscope show expression pattern of V-ATPase ‘a2' subunit isoform (a2V in red). Nucleus was stained with DAPI (diamidino-2-phenylindole; blue). Scale bars: 20 μ m
Figure Legend Snippet: a2V expression during normal mammary development. Inguinal mammary glands from puberty, virginity, pregnancy, lactation and involution stages were harvested and analyzed for gene expression and cellular distribution of a2V. Total RNA extracted from mammary glands was subjected to quantitative real-time-PCR (qRT-PCR) analysis. ( a ) Gene expression of V-ATPase ‘a2' subunit isoform during various stages of gland development compared with puberty. ( b ) Relative gene expression of V-ATPase a1, a2 and a3 subunit isoforms compared with each other at every stage. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as endogenous housekeeping control. Data represent mean±S.E., n =6. * P ≤0.05, ** P ≤0.01, *** P ≤0.001 and **** P ≤0.0001. ( c ) Representative z-stack images from a confocal laser-scanning microscope show expression pattern of V-ATPase ‘a2' subunit isoform (a2V in red). Nucleus was stained with DAPI (diamidino-2-phenylindole; blue). Scale bars: 20 μ m

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Laser-Scanning Microscopy, Staining

4) Product Images from "A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface"

Article Title: A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.161282998

Indirect immunofluorescence localization of Pma1-10. Cells were shifted to 37°C for 3 h, and then fixed, permeabilized, and stained with polyclonal anti-Pma1 and monoclonal anti-V-ATPase antibodies. ( A ) Pma1 localization in wild-type (L3852), pma1 - 10 end4–1 (XGX42–8B), pma1 - 10 (XGX28–1C), and pma1 - 10 pep4Δ (XGX28–1A) cells. Colocalization of Pma1 and V-ATPase at the vacuolar membrane is seen in pma1 - 10 pep4 cells. ( B ) Double labeling with anti-Pma1 and anti-V-ATPase in pma1 - 10 vps27Δ (XGX47–1D). Pma1 colocalizes in a large punctate spot with V-ATPase.
Figure Legend Snippet: Indirect immunofluorescence localization of Pma1-10. Cells were shifted to 37°C for 3 h, and then fixed, permeabilized, and stained with polyclonal anti-Pma1 and monoclonal anti-V-ATPase antibodies. ( A ) Pma1 localization in wild-type (L3852), pma1 - 10 end4–1 (XGX42–8B), pma1 - 10 (XGX28–1C), and pma1 - 10 pep4Δ (XGX28–1A) cells. Colocalization of Pma1 and V-ATPase at the vacuolar membrane is seen in pma1 - 10 pep4 cells. ( B ) Double labeling with anti-Pma1 and anti-V-ATPase in pma1 - 10 vps27Δ (XGX47–1D). Pma1 colocalizes in a large punctate spot with V-ATPase.

Techniques Used: Immunofluorescence, Staining, Labeling

5) Product Images from "Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant "

Article Title: Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

Journal: The Journal of Cell Biology

doi:

Indirect immunofluorescence localization of mutant Pma1 in a prevacuolar compartment. Localization of Pma1 and V-ATPase in pma1-7 and pma1-7 vps27 ts cells. Mid-log cultures were harvested, fixed, and spheroplasted. Cells were permeabilized with SDS before staining with rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kD subunit of V-ATPase, followed by species-specific CY3- and DTAF-conjugated secondary antibodies. ( Upper panel ) Mutant Pma1 staining is coincident with V-ATPase staining at the vacuolar membrane in pma1-7 cells at 30°C. In these cells, mutant Pma1 is also seen as spots distinct from the vacuole ( arrowheads ). Nomarski optics show vacuoles as indentations. ( Lower panel ) pma1-7 vps27 ts cells were shifted to 37°C for 1 h. Both mutant Pma1 and the 60-kD V-ATPase subunit are accumulated in the prevacuolar compartment.
Figure Legend Snippet: Indirect immunofluorescence localization of mutant Pma1 in a prevacuolar compartment. Localization of Pma1 and V-ATPase in pma1-7 and pma1-7 vps27 ts cells. Mid-log cultures were harvested, fixed, and spheroplasted. Cells were permeabilized with SDS before staining with rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kD subunit of V-ATPase, followed by species-specific CY3- and DTAF-conjugated secondary antibodies. ( Upper panel ) Mutant Pma1 staining is coincident with V-ATPase staining at the vacuolar membrane in pma1-7 cells at 30°C. In these cells, mutant Pma1 is also seen as spots distinct from the vacuole ( arrowheads ). Nomarski optics show vacuoles as indentations. ( Lower panel ) pma1-7 vps27 ts cells were shifted to 37°C for 1 h. Both mutant Pma1 and the 60-kD V-ATPase subunit are accumulated in the prevacuolar compartment.

Techniques Used: Immunofluorescence, Mutagenesis, Staining

Related Articles

Transfection:

Article Title: A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo
Article Snippet: .. For vacuolation test, SNX10-GFP was transfected into Hela cells pre-treated with siRNAs to V-ATPase or a control siRNA using the Lipofectamine 2000 reagent according to the manufacturer's protocol (Invitrogen). .. For drug treatment, Bafilomycin A1 (10 nM, Sigma) was added to cells 8 h after transfection.

Infection:

Article Title: Mycobacterium leprae Phenolglycolipid-1 Expressed by Engineered M. bovis BCG Modulates Early Interaction with Human Phagocytes
Article Snippet: .. LysoTracker Red labelling was performed by washing hMDM at different time points after infection and incubating them with the acidotropic dye (1∶2000) in RPMI 1640 for 1 h. Rinsed cells were fixed with 3.7% paraformaldehyde for 1 h. For v-ATPase or CD63, mouse monoclonal Ab against human CD63 and rabbit polyclonal anti-serum against v-ATPase proton pump were obtained from Caltag Laboratories (Burlingame, USA) and from Synaptic Systems (Göttingen, Germany), respectively. .. Macrophages were fixed as described above, permeabilized by incubation with 0.3% Triton X-100 for 10 minutes at room temperature (RT), blocked by incubation with 0.3% BSA and incubated with antiserum against v-ATPase (1/100) or mouse anti-CD63 Ab (1∶100) for 1 h at RT, and revealed with Rhodamine-Red conjugated goat anti-rabbit or anti-mouse Ab.

Microarray:

Article Title: V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration
Article Snippet: .. V-ATPase is specifically upregulated during adult caudal fin regeneration To find candidate H+ transporters that could be associated to regeneration and to the detected H+ efflux, we took advantage of an Affymetrix microarray previously done in our laboratory to assess gene expression during regeneration after distal amputation. .. Upon a specific analysis for proton transporters' expression, we focused on the V-ATPase, one of the main H+ transporters in animal cells.

Incubation:

Article Title: Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant
Article Snippet: .. For double staining of Pma1 and vacuolar H+ -ATPase, cells were permeabilized with SDS, as described , followed by incubation with rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kD subunit of the V-ATPase (Molecular Probes, Inc., Eugene, OR). .. Primary antibody staining was visualized with Cy3- and DTAF-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA).

Expressing:

Article Title: The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling
Article Snippet: .. Prevalidated Taqman Gene expression primers for V-ATPase ‘a' subunit isoforms ATP6V0a1 , ATP6V0a2 , ATP6V0a3 and ATP6V0a4 ; Notch pathway genes Notch 1 , Hey1 ; TGF-β1 , pSmad2 , mammary proliferation markers Areg , Cited 1 , lactation marker β -casein and internal control GAPDH and 18srRNA were all purchased from Applied Biosystems. .. Universal Fast PCR Master Mix Reagent (Applied Biosystems, Thermo Fisher Scientific) was used for qPCR amplification of the cDNA.

Article Title: V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration
Article Snippet: .. V-ATPase is specifically upregulated during adult caudal fin regeneration To find candidate H+ transporters that could be associated to regeneration and to the detected H+ efflux, we took advantage of an Affymetrix microarray previously done in our laboratory to assess gene expression during regeneration after distal amputation. .. Upon a specific analysis for proton transporters' expression, we focused on the V-ATPase, one of the main H+ transporters in animal cells.

Marker:

Article Title: The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling
Article Snippet: .. Prevalidated Taqman Gene expression primers for V-ATPase ‘a' subunit isoforms ATP6V0a1 , ATP6V0a2 , ATP6V0a3 and ATP6V0a4 ; Notch pathway genes Notch 1 , Hey1 ; TGF-β1 , pSmad2 , mammary proliferation markers Areg , Cited 1 , lactation marker β -casein and internal control GAPDH and 18srRNA were all purchased from Applied Biosystems. .. Universal Fast PCR Master Mix Reagent (Applied Biosystems, Thermo Fisher Scientific) was used for qPCR amplification of the cDNA.

Staining:

Article Title: A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface
Article Snippet: .. Cells were spheroplasted and permeabilized with methanol and acetone before staining with affinity-purified rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kDa subunit of V-ATPase (Molecular Probes). .. Primary antibody staining was detected with Cy3- and Cy2-conjugated secondary antibodies (Jackson ImmunoResearch).

Article Title: Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1
Article Snippet: .. Cells were spheroplasted and permeabilized with methanol and acetone before staining with affinity-purified rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kDa subunit of V-ATPase (Molecular Probes). .. Primary antibody staining was detected with Cy3- and Cy2-conjugated secondary antibodies (Jackson ImmunoResearch).

Affinity Purification:

Article Title: A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface
Article Snippet: .. Cells were spheroplasted and permeabilized with methanol and acetone before staining with affinity-purified rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kDa subunit of V-ATPase (Molecular Probes). .. Primary antibody staining was detected with Cy3- and Cy2-conjugated secondary antibodies (Jackson ImmunoResearch).

Article Title: Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1
Article Snippet: .. Cells were spheroplasted and permeabilized with methanol and acetone before staining with affinity-purified rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kDa subunit of V-ATPase (Molecular Probes). .. Primary antibody staining was detected with Cy3- and Cy2-conjugated secondary antibodies (Jackson ImmunoResearch).

Double Staining:

Article Title: Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant
Article Snippet: .. For double staining of Pma1 and vacuolar H+ -ATPase, cells were permeabilized with SDS, as described , followed by incubation with rabbit anti-Pma1 and mouse monoclonal antibody against the 60-kD subunit of the V-ATPase (Molecular Probes, Inc., Eugene, OR). .. Primary antibody staining was visualized with Cy3- and DTAF-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA).

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    Thermo Fisher v atpase
    Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, <t>V-ATPase</t> H and <t>SV2a</t> in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p
    V Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher na k atpase monoclonal antibody
    RT-PCR analysis of <t>Na,K-ATPase</t> ( α 1 subunit), MRP4, NaDC1, NaDC3, OAT1, and OAT3 in human ciliary body (CB), retina (Ret), iris, retinal pigmented epithelium (RPE), and cornea (Corn). Kidney was used as a positive control. Polymerase chain reaction products were separated on 1% agarose gels and visualized with ethidium bromide. + con, positive control.
    Na K Atpase Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher v atpase v0a1
    RT-PCR analysis of <t>Na,K-ATPase</t> ( α 1 subunit), MRP4, NaDC1, NaDC3, OAT1, and OAT3 in human ciliary body (CB), retina (Ret), iris, retinal pigmented epithelium (RPE), and cornea (Corn). Kidney was used as a positive control. Polymerase chain reaction products were separated on 1% agarose gels and visualized with ethidium bromide. + con, positive control.
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    Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p

    Journal: Scientific Reports

    Article Title: Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson’s disease

    doi: 10.1038/srep40887

    Figure Lengend Snippet: Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p

    Article Snippet: COX IV (1:2000, Abcam #ab16056, 16 kD), NDUSF4 (1:500, Santa-Cruz #sc-100567, 18 kD), TH (1:2000, Millipore #MAB318, 58 kD), Actin (1:500, Santa-Cruz #sc-1615, 43 kD), Synaptophysin (1:2000, Cell Signaling #D35E4, 38 kD), V-ATPase (1:1000, ThermoFisher #PA5-22134, 55 kD), SV2a (1:1000, Santa-Cruz #sc-11936, 93 kD) were the primary antibodies used.

    Techniques: Mutagenesis, Expressing, Mouse Assay

    RT-PCR analysis of Na,K-ATPase ( α 1 subunit), MRP4, NaDC1, NaDC3, OAT1, and OAT3 in human ciliary body (CB), retina (Ret), iris, retinal pigmented epithelium (RPE), and cornea (Corn). Kidney was used as a positive control. Polymerase chain reaction products were separated on 1% agarose gels and visualized with ethidium bromide. + con, positive control.

    Journal: Molecular Pharmacology

    Article Title: A Renal-Like Organic Anion Transport System in the Ciliary Epithelium of the Bovine and Human Eye

    doi: 10.1124/mol.114.096578

    Figure Lengend Snippet: RT-PCR analysis of Na,K-ATPase ( α 1 subunit), MRP4, NaDC1, NaDC3, OAT1, and OAT3 in human ciliary body (CB), retina (Ret), iris, retinal pigmented epithelium (RPE), and cornea (Corn). Kidney was used as a positive control. Polymerase chain reaction products were separated on 1% agarose gels and visualized with ethidium bromide. + con, positive control.

    Article Snippet: The mouse anti- α 1 subunit of Na,K-ATPase monoclonal antibody (clone M8-P1-A3) was obtained from Thermo Scientific (Rockford, IL).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction

    Histopathology and immunofluorescence in CASQ-1 myopathy Serial cryosections of p.Asp244Gly (patient 8) and p.Gly103Asp (patient 14) muscle biopsy (all panels original magnification ×20). In patient 8, in hematoxylin-eosin (H E) optically empty vacuoles almost exclusively in type II fibers, increased fiber size variation, central nuclei, and splitting are shown. Immunofluorescence analysis of sarcoplasmic Ca 2+ influx/efflux machinery proteins shows reactivity for calsequestrin 1 (CASQ1), ORAI1, SERCA1, and STIM1 at the edge of the vacuoles that appear optically empty with acidic ATPase. In patient 14, vacuoles filled with amorphous material (H E) corresponding to tubular aggregates are shown. No optically empty vacuoles are detected. The intrafiber vacuoles are strongly immunoreactive for CASQ1, ORAI1, SERCA1, and STIM1 and weakly immunoreactive for SERCA2.

    Journal: Neurology

    Article Title: The clinical spectrum of CASQ1-related myopathy

    doi: 10.1212/WNL.0000000000006387

    Figure Lengend Snippet: Histopathology and immunofluorescence in CASQ-1 myopathy Serial cryosections of p.Asp244Gly (patient 8) and p.Gly103Asp (patient 14) muscle biopsy (all panels original magnification ×20). In patient 8, in hematoxylin-eosin (H E) optically empty vacuoles almost exclusively in type II fibers, increased fiber size variation, central nuclei, and splitting are shown. Immunofluorescence analysis of sarcoplasmic Ca 2+ influx/efflux machinery proteins shows reactivity for calsequestrin 1 (CASQ1), ORAI1, SERCA1, and STIM1 at the edge of the vacuoles that appear optically empty with acidic ATPase. In patient 14, vacuoles filled with amorphous material (H E) corresponding to tubular aggregates are shown. No optically empty vacuoles are detected. The intrafiber vacuoles are strongly immunoreactive for CASQ1, ORAI1, SERCA1, and STIM1 and weakly immunoreactive for SERCA2.

    Article Snippet: Serial cryosections of muscle biopsies were stained with hematoxylin-eosin, ATPase, nicotinamide adenine dinucleotide (NADH), and mouse monoclonal antibodies against calsequestrin (clone VIIID12; Thermo Fisher, Waltham, MA), ryanodine receptor, RYR1 (clone 34C; Abcam, Cambridge, UK), sarco/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1; clone VE121G9; Santa Cruz Biotechnology, Santa Cruz, CA), stromal interaction molecule 1 (STIM1; clone 44/GOK; BD, San Jose, CA), rabbit polyclonal calcium release-activated calcium modulator 1, (ORAI1; Sigma-Aldrich Srl, Milan, Italy), and goat polyclonal sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2; N19; Santa Cruz).

    Techniques: Histopathology, Immunofluorescence