MA3-923

antibody from Invitrogen Antibodies

Targeting: ATP4B ATP6B

Provider product page for MA3-923

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [67]
Comments [0]
Validations
Immunocytochemistry [3]
Immunohistochemistry [2]
Other assay [14]

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Product number: MA3-923 - Provider product page
Provider: Invitrogen Antibodies
Product name: ATP4B Monoclonal Antibody (2G11)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA3-923 detects the beta-subunit of hydrogen/potassium ATPase from bovine, human, canine, porcine, rabbit, mouse, ferret, and rat tissues. MA3-923 has been successfully used in Western blot, immunohistochemistry, immunocytochemistry, and immunoprecipitation procedures (ChIP). By Western blot, this antibody detects various forms of the beta-subunit, including a 60 to 80 kDa glycosylated protein, a 52 kDa beta-subunit precursor, and the 34 kDa core peptide from rabbit stomach extracts. Immunohistochemical staining of hydrogen/potassium ATPase in rabbit gastric glands with MA3-923 results in specific staining of parietal cells. MA3-923 has been shown to inhibit the enzymatic activity of the hydrogen/potassium ATPase and to alter the affinity of the cytoplasmic potassium binding site. The MA3-923 antigen is purified 34 kDa core peptide from deglycosylated hog gastric microsomes. This antibody recognizes an epitope between amino acid residues 1-13 or 15-28 located on the cytoplasmic side of the beta-subunit.
Reactivity: Human, Mouse, Rat, Bovine, Canine, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: 2G11
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

ATP4B protein structure - MA3-923 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Hydrogen/Potassium ATPase beta using Anti-Hydrogen/Potassium ATPase beta Monoclonal Antibody (2G11) (Product # MA3-923) shows staining in A2058 Cells. Hydrogen/Potassium ATPase beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Hydrogen/Potassium ATPase beta (Product # MA3-923) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Hydrogen/Potassium ATPase beta using Anti-Hydrogen/Potassium ATPase beta Monoclonal Antibody (2G11) (Product # MA3-923) shows staining in Hela Cells. Hydrogen/Potassium ATPase beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Hydrogen/Potassium ATPase beta (Product # MA3-923) at a dilution of 1:200 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Hydrogen/Potassium ATPase beta using Anti-Hydrogen/Potassium ATPase beta Monoclonal Antibody (2G11) (Product # MA3-923) shows staining in U251 Cells. Hydrogen/Potassium ATPase beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Hydrogen/Potassium ATPase beta (Product # MA3-923) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human gastric carcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Hydrogen/Potassium ATPase beta (Product # MA3-923) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human stomach tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Hydrogen/Potassium ATPase beta (Product # MA3-923) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 10 Organoid-derived SPEM cell lineages may contribute to wound healing in the aged stomach. ( A ) Immunofluorescence staining of ulcerated gastric tissue of aged mice transplanted with gastric organoids 30 days after injury. A lectin specific for surface mucous pit cells (UEAI, green) and antibodies specific for parietal cells (HK, red) and endocrine cells (CgA, blue) were used to identify cell lineages. Scale bars : 50 mum. ( B ) Quantification of UEAI+, HK+, and CgA+ cells within the damaged epithelium of aged mice transplanted with gastric organoids 1-30 days after injury. Data are presented as means +- SEM, N = 3-4 mice per time point. * P < .05 compared with day 0, determined by 1-way analysis of variance. ( C ) Ulcer measurements from young (black), aged (red), and aged mice transplanted with gastric organoids (green) at 1, 3, and 7 days after injury. Data are expressed as means +- SEM. * P < .05 compared with aged transplanted with organoids using 1-way analysis of variance, N = 3-10 mice per time point. ( D ) Ulcer measurements from young mice without (W/O) or with (W/) organoid transplantation at 1, 3, 7, and 30 days after injury. Data are expressed as means +- SEM. * P < .05 compared with young untransplanted mice using 1-way analysis of variance, N = 4 mice per time point. Whole mount immunofluorescence staining to identify parietal cells using antibody specific for H+K+-adenosine triphosphatase in stomach tissue collected from young mice without or with org

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 YFP expression in gastric epithelium of organoid transplanted mice. ( A ) Immunofluorescence staining using an antibody against YFP in untransplanted mice 7 days after injury. Scale bars : 50 mum. ( B ) YFP expression by RT-PCR using RNA prepared from laser capture microdissection of ulcerated and intact epithelium from stomachs 7 days after injury and gastric organoid transplantation. CAP, captured tissue. ( C ) Single cells flow sorted from stomach tissue of aged transplanted mice for YFP+ and YFP- grown in culture for 10 days. ( D ) Fluorescence-activated cell sorting (FACS) histogram depicting YFP+ single-cell population isolated from gastric tissue of aged mice transplanted with gastric organoids. YFP+ single cells were collected from the gastric glands 5 months after injury and gastric organoid transplantation and used to generate YFP+ gastric organoids in culture.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Identification of different cell types expressing GATA-4 and GATA-6 proteins in normal gastrointestinal mucosa . Staining for chromogranin A (ChrA) (blue-greyish cytoplasmic staining in A, A', C, C', F, and F') was used to detect neuroendocrine cells and staining for H+/K+-ATPase alpha (HK) (blue-greyish cytoplasmic staining in B and B' to detect parietal cells. The samples were double-stained for GATA-4 (G4) or GATA-6 (G6) (brown nuclear staining). The GATA proteins are not expressed in the neuroendocrine cells of the stomach, duodenum, or colon (A, A', C, C', F and F' respectively; arrowheads). GATA-4 is detected in the H+/K+-ATPase alpha presenting parietal cells (B). GATA-6 is positive in approximately two thirds of the parietal cells positive for H+/K+-ATPase alpha (B', arrowheads). Some duodenal goblet cells, stained deep blue by the Alcian blue method (Alc) (D'), and most Paneth cells are positive for GATA-6 (E', arrowhead) showing no immunoreactivity for GATA-4 (D and E, arrowheads). Scale bar = 50 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Human gastric organoids contain differentiated antral cell types a , Schematic representation of a typical antral gland showing normal cell types and associated molecular markers. b-g , Immunofluorescent staining demonstrated that 34-day hGOs consisted of normal cell types found in the antrum, but not the fundus. The hGO epithelium contained surface mucous cells that express MUC5AC ( b1, b1' ), similar to the postnatal day 12 (P12) mouse antrum ( b2, b2' ), but not ATP4B-expressing parietal cells ( c1, c1' ) that characterize the fundus ( c2, c2' ). SOX9 + cells were found at the base of the hGO epithelium ( d1, d1' ), similar to the progenitor cells found in the P12 antrum ( d2, d2' ). Further, hGOs contained MUC6 + antral gland cells ( e ) and LGR5 -expressing cells ( f ). Boxed regions in b-f are shown as high magnification images indicated by an apostrophe ('). g, Day 34 hGOs also contained endocrine cells (SYP) that expressed the gastric hormones GAST, SST, GHRL, and serotonin (5-HT). Scale bars, 100 mum in b-f , 20 mum in b'-f' and g . Marker expression data are representative from a minimum of 10 independent experiments, except LGR5-GFP data, which is a representative example from two separate experiments.

Supportive validation

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Experimental details: 4 Photomicrographs of double stained formalin-fixed, dispersed glands from the oxyntic mucosa, showing somatostatin-immunolabelled D-cells (stained red) and H + /K + ATPase-immunolabelled parietal cells (stained black). No connections between D-cells and parietal cells were seen. The cytoplasmic elongations were connected to other nonstained mucosal cells. For the double-staining technique, the cell smears were washed for 2 h in a glycine-HCL buffer (pH 2.2) between antibodies. Monoclonal Anti H + /K + ATPase Beta was used at a dilution of 1 : 2000 as a marker of parietal cells (MA3-923; Affinity Bioreagents, Golden, CO, USA). AEC peroxidase kit (SK4200; Vector) and a SG peroxidase kit (SK4700; Vector) were used to visualise the immunoreactions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 3 FIGURE Possible involvement of H + /K + -ATPase in cAMP effects on lysosomal acidification: siRNAs. (a) Expression of H + /K + -ATPase in astrocytes and siRNA efficiency to Atp4a or Atp4b. RNA levels (upper panels) and protein levels (middle panels) of the subunits of H + /K + -ATPase (Atp4a and Atp4b) and Atp6 in astrocytes treated with scramble siRNA or siRNA for Atp4a or Atp4b for 72 hr. Expression levels of each RNAs were represented as a relative fold compared to those of cells treated with scramble siRNA (lower panels) ( n = 4, mean +- SEM, Student's t -test; *** p < .001). (b) Confocal photomicrographs of DND-189-loaded Atp4a or Atp4b knockdown (KD) astrocytes before and 1 hr after treatment with BafA1 or BafA1 plus cAMP (BafA1/cAMP). Cortical astrocytes were transfected with scramble siRNA or siRNA to Atp4a or Atp4b for 72 hr and DND-189 fluorescence changes in live cells were examined under a confocal microscope. The H + /K + -ATPase subunit-targeting siAtp4a or siAtp4b did not significantly affect lysosomal alkalization by BafA1 alone (left panels). In contrast, siRNA-mediated Atp4a or Atp4b KD attenuated the cAMP-mediated reversal of the effects of BafA1 (middle panels) (scale bar, 20 mum). Fluorescence changes in DND-189 were analyzed by the ""Mean ROI"" option in Zen software ( n = 3-9, mean +- SEM). * p < .05, ** p < .01, *** p < .001 versus control cells (one-way analysis of variance [ANOVA], one-tailed t -test); n.s., not significant, ### p < .001 (Kruskal-W

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 6 FIGURE Increased association between H + /K + -ATPase and ZnT3 and their lysosomal recruitments by cAMP. (a) Immunoblotting for ATP4B and ZnT3-HA in anti-HA or anti-ATP4B antibody immunoprecipitates prepared from astrocytes transfected with ZnT3-HA for 72 hr. ATP4B was co-immunoprecipitated with ZnT3-HA (upper panels) and ZnT3-HA was co-immunoprecipitated with ATP4B (lower panels). Arrowheads show specific signals detected by each antibody and asterisk indicates the light chain of immunoglobulin. Experiments were repeated at least five times and representative images were shown. (b) Immunoblotting for ATP4B and ZnT3 in anti-ZnT3 or anti-ATP4B antibody immunoprecipitates in samples prepared from WT and ZnT3- (KO) astrocytes. ATP4B or ZnT3 was bound to each other at the cellular endogenous level. Arrowheads show specific signals detected by each antibody and asterisk indicates nonspecific bands (left panels, Input) or heavy chain of immunoglobulin (middle and right panels, IP). Experiments were repeated at least three times and representative images were shown. (c) Immunoblotting for ZnT3-HA and ATP4B in anti-HA or anti-ATP4B antibody immunoprecipitates from ZnT3-HA-transfected astrocytes after sham wash (CTL) or a 6-hr treatment with BafA1 alone or BafA1 plus cAMP. The addition of cAMP enhanced the association between H + /K + -ATPase and ZnT3. Arrowheads show ATP4B or HA by each antibody and asterisk indicates nonspecific bands (in input), light chain of immunoglobulin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 Comparison of gene expression between two subtypes of EPN-PF. a Representative photomicrographs stained with ATP4B. The expression of ATP4B in EPN-PFB was significantly higher than that in EPN-PFA ( P = 0.0363). b Representative photomicrographs stained with CCDC151. The expression of CCDC151 in EPN-PFB was significantly higher than that in EPN-PFA ( P = 0.0280). c Representative photomicrographs stained with DMKN. The expression of DMKN in EPN-PFB was significantly higher than that in EPN-PFA ( P = 0.0003). d Representative photomicrographs stained with SCN4B.The expression of SCN4B in EPN-PFB was higher than that in EPN-PF, but the difference was no statistically significant ( P = 0.0363)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Blood estrogen levels and production of gastric estrogen are directly regulated by blood triglyceride levels in males. a Male rats (8 weeks old, deprived of food for 4 h) were intravenously injected with soy oil emulsion (2 mL of 20 % soy oil emulsion per kg body weight, n = 10) or control saline (control, n = 10). TG and E2 levels in the tail venous blood were measured before (0) and at 0.1, 0.5, 1, 1.5, 2, 3, 4, and 5 h after the injection. b Isolated gastric gland epithelium from male rats. c Gastric mucosa of the male rat was double-stained for GPR120 (G-protein-coupled receptor 120) or CD36 (cluster of differentiation 36) (green) with ATP4B (magenta). d Isolated epithelium from male rat (deprived of food for 4 h) was incubated in DMEM with or without testosterone (20 nM) or lauric acid (500 uM) at 37 degC for 1 h. E2 levels, normalized by phospholipid, were compared to those incubated with 20 nM testosterone but without lauric acid ( n = 9). n number of rats. Data were mean +- s.d. a (0.5 h) and d , P values determined by two-sided Student's t -test. Bars: (main panels) 50 um; (insets) 10 um.

Supportive validation

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Experimental details: Fig. 5 Stomach of OVX female rats, like of male rats, secretes estrogen in response to the blood triglyceride levels. a Gastric mucosa from an OVX female rat (8 weeks old, operated 14 days before) was double-stained for ACADM or GCK (green) with aromatase [arom] or ATP4B (magenta). b OVX female rats, deprived of food for 4 h, were orally administered olive oil (2.5 mL per kg body weight, n = 9) or control water ( n = 9). TG and E2 levels in the tail venous blood measured before (0) and at 1, 2, 3, 4, and 5 h after the administration (left), and the correlation diagram between the blood TG and E2 levels at 2 h after the administration ( n = 18, right). c Isolated epithelium from OVX female rat (deprived of food for 4 h) was incubated in DMEM with or without testosterone (20 nM) or lauric acid (500 uM) at 37 degC for 1 h. E2 levels, normalized by phospholipid, were compared to those incubated with 20 nM testosterone but without lauric acid ( n = 10). n number of rats. Data were mean +- s.d. b left (2 h) and c P values determined by two-sided Student's t -test. b right, R and P values determined by Pearson's product-moment correlation with 95% density ellipse. Bars: (main panels) 50 um; (insets) 10 um.