Structured Review

Austral Biologicals anti caga
<t>CagA</t> is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and <t>VacA</t> respectively.
Anti Caga, supplied by Austral Biologicals, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Biochemical and functional characterization of Helicobacter pylori vesicles"

Article Title: Biochemical and functional characterization of Helicobacter pylori vesicles

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2010.07307.x

CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.
Figure Legend Snippet: CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.

Techniques Used:

Identification of major protein components in H. pylori vesicles. A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane. B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified. C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis. D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots. E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left. F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.
Figure Legend Snippet: Identification of major protein components in H. pylori vesicles. A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane. B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified. C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis. D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots. E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left. F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.

Techniques Used: SDS Page, Mass Spectrometry, Functional Assay, Expressing, Western Blot

2) Product Images from "Regulation of p53 tumor suppressor by Helicobacter pylori in gastric epithelial cells"

Article Title: Regulation of p53 tumor suppressor by Helicobacter pylori in gastric epithelial cells

Journal: Gastroenterology

doi: 10.1053/j.gastro.2010.06.018

CagA regulates p53 levels in H. pylori -infected cells (A) AGS cells were cultured in the presence of the wild-type H. pylori strain 7.13 or isogenic cagA− or cagE− null mutants, and protein levels of p53 were assessed by Western blotting. (B) Left panel: gastric tissues harvested from gerbils infected with H. pylori strain 7.13 or isogenic cagA- null mutant at indicated time points were immunostained for p53 and quantitated using a blind protocol. Results are expressed as the percentage of p53 positive cells per sample. Mean values ( ) for cagA − and cagA + infected animals are shown. A dashed line depicts the average levels of p53 in the uninfected control animals. Right panel: representative immunohistochemical staining for p53 (x20) is shown for uninfected animals (1) and those infected with wild-type (2) or cagA − (3) isogenic H. pylori strains for 6 hours (at the peak of p53 increase). Levels of p53 were also analyzed by Western blotting with p53-specific antibody at 6 hours. (C) Analysis of HDM2 phosphorylation in AGS cells co-cultured with H. pylori strain J166 or its isogenic cagA− or cagE− derivatives. (D) Analysis of p53 ubiquitination in AGS cells co-cultured with the indicated isogenic H. pylori strains for 24 hours. Proteasomal degradation was inhibited with MG-132.
Figure Legend Snippet: CagA regulates p53 levels in H. pylori -infected cells (A) AGS cells were cultured in the presence of the wild-type H. pylori strain 7.13 or isogenic cagA− or cagE− null mutants, and protein levels of p53 were assessed by Western blotting. (B) Left panel: gastric tissues harvested from gerbils infected with H. pylori strain 7.13 or isogenic cagA- null mutant at indicated time points were immunostained for p53 and quantitated using a blind protocol. Results are expressed as the percentage of p53 positive cells per sample. Mean values ( ) for cagA − and cagA + infected animals are shown. A dashed line depicts the average levels of p53 in the uninfected control animals. Right panel: representative immunohistochemical staining for p53 (x20) is shown for uninfected animals (1) and those infected with wild-type (2) or cagA − (3) isogenic H. pylori strains for 6 hours (at the peak of p53 increase). Levels of p53 were also analyzed by Western blotting with p53-specific antibody at 6 hours. (C) Analysis of HDM2 phosphorylation in AGS cells co-cultured with H. pylori strain J166 or its isogenic cagA− or cagE− derivatives. (D) Analysis of p53 ubiquitination in AGS cells co-cultured with the indicated isogenic H. pylori strains for 24 hours. Proteasomal degradation was inhibited with MG-132.

Techniques Used: Infection, Cell Culture, Western Blot, Mutagenesis, Immunohistochemistry, Staining

CagA induces degradation of p53 (A) Left panel: p53-null Kato III cells were co-transfected with the indicated plasmids and GFP for 48 hours and then analyzed for p53 expression. Gel loading was normalized to GFP expression. HDM2 and p53 co-transfection was used as an additional positive control. Right panel: the same as the left panel but another p53-null osteosarcoma cell line, SaOs2, was used. (B) AGS cells were transfected with CagA-IRES-GFP (CagA) or empty IRES-GFP (Control) vectors. Twenty-four hours post-transfection cells were analyzed by immunofluorescence for p53 (red) in GFP-expressing cells (green). Nuclear p53 protein disappeared in 58% of CagA-expressing cells whereas only 13.5% of control GFP-positive cells were negative for p53. (C) Left panel: AGS cells that express CagA under control of tetracycline-inducible promoter were treated with hydrogen peroxide or left untreated and then analyzed for p53. Right panel: Control AGS cells (−DOX) or ones expressing CagA (+DOX) were treated with indicated concentrations of H 2 O 2 for 24 hours. Cell death was assessed by flow cytometry after propidium iodide staining. The proportion of cells in subG1 is shown. CagA significantly increased survival of cells treated with H 2 O 2 . **, p
Figure Legend Snippet: CagA induces degradation of p53 (A) Left panel: p53-null Kato III cells were co-transfected with the indicated plasmids and GFP for 48 hours and then analyzed for p53 expression. Gel loading was normalized to GFP expression. HDM2 and p53 co-transfection was used as an additional positive control. Right panel: the same as the left panel but another p53-null osteosarcoma cell line, SaOs2, was used. (B) AGS cells were transfected with CagA-IRES-GFP (CagA) or empty IRES-GFP (Control) vectors. Twenty-four hours post-transfection cells were analyzed by immunofluorescence for p53 (red) in GFP-expressing cells (green). Nuclear p53 protein disappeared in 58% of CagA-expressing cells whereas only 13.5% of control GFP-positive cells were negative for p53. (C) Left panel: AGS cells that express CagA under control of tetracycline-inducible promoter were treated with hydrogen peroxide or left untreated and then analyzed for p53. Right panel: Control AGS cells (−DOX) or ones expressing CagA (+DOX) were treated with indicated concentrations of H 2 O 2 for 24 hours. Cell death was assessed by flow cytometry after propidium iodide staining. The proportion of cells in subG1 is shown. CagA significantly increased survival of cells treated with H 2 O 2 . **, p

Techniques Used: Transfection, Expressing, Cotransfection, Positive Control, Immunofluorescence, Flow Cytometry, Cytometry, Staining

3) Product Images from "Biochemical and functional characterization of Helicobacter pylori vesicles"

Article Title: Biochemical and functional characterization of Helicobacter pylori vesicles

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2010.07307.x

CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.
Figure Legend Snippet: CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.

Techniques Used:

Identification of major protein components in H. pylori vesicles. A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane. B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified. C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis. D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots. E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left. F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.
Figure Legend Snippet: Identification of major protein components in H. pylori vesicles. A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane. B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified. C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis. D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots. E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left. F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.

Techniques Used: SDS Page, Mass Spectrometry, Functional Assay, Expressing, Western Blot

4) Product Images from "Conversion of Helicobacter pylori CagA from senescence inducer to oncogenic driver through polarity-dependent regulation of p21"

Article Title: Conversion of Helicobacter pylori CagA from senescence inducer to oncogenic driver through polarity-dependent regulation of p21

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20100602

Involvement of the ROCK–c-Myc pathway in the suppression of p21 upon expression of CagA in polarized epithelial cells. (A, left) Polarized MDCK cells were transfected with indicated vectors. At 6 h after transfection, cells were treated with 50 µM Y-27632 and were cultured for additional 21 h. Cells were then stained with anti-p21 antibody (red), anti-HA antibody (green), and DAPI (blue). Confocal x-y images are shown. Bar, 10 µm. (A, right) Percentage of p21-positive cells in MDCK cells expressing EGFP or CagA. Error bars indicate mean ± SD. n = 3. **, P
Figure Legend Snippet: Involvement of the ROCK–c-Myc pathway in the suppression of p21 upon expression of CagA in polarized epithelial cells. (A, left) Polarized MDCK cells were transfected with indicated vectors. At 6 h after transfection, cells were treated with 50 µM Y-27632 and were cultured for additional 21 h. Cells were then stained with anti-p21 antibody (red), anti-HA antibody (green), and DAPI (blue). Confocal x-y images are shown. Bar, 10 µm. (A, right) Percentage of p21-positive cells in MDCK cells expressing EGFP or CagA. Error bars indicate mean ± SD. n = 3. **, P

Techniques Used: Expressing, Transfection, Cell Culture, Staining

Induction of p21-targeting microRNAs upon expression of CagA in polarized epithelial cells. (A) Polarized MDCK cells were infected with adenovirus transducing CagA or β-galactosidase (control). At 6 h after infection, cells were treated with or without Y-27632 at a final concentration of 50 µM and were cultured for an additional 18 h. Expression of miR-17 (left) or miR-20a (right) was quantified by real-time PCR. Error bars indicate mean ± SD. n = 3 for miR-17, n = 4 for miR-20a. **, P
Figure Legend Snippet: Induction of p21-targeting microRNAs upon expression of CagA in polarized epithelial cells. (A) Polarized MDCK cells were infected with adenovirus transducing CagA or β-galactosidase (control). At 6 h after infection, cells were treated with or without Y-27632 at a final concentration of 50 µM and were cultured for an additional 18 h. Expression of miR-17 (left) or miR-20a (right) was quantified by real-time PCR. Error bars indicate mean ± SD. n = 3 for miR-17, n = 4 for miR-20a. **, P

Techniques Used: Expressing, Infection, Concentration Assay, Cell Culture, Real-time Polymerase Chain Reaction

Induction of forced mitogenesis upon expression of CagA in polarized epithelial cells. (A) After expression of HA-tagged CagA in polarized MDCK cells, CagA-positive cells were visualized with anti-HA antibody (green). Polarized MDCK cells without CagA expression were used as a control. Nuclei in confocal x-z images were visualized by 4,6-diamidino-2-phenylindole (DAPI) staining (blue). Bars, 10 µm. (B, left) Polarized MDCK cells transfected with an HA-tagged CagA vector were stained with anti-HA antibody (green), anti-p21 antibody (red), and DAPI (blue). Confocal x-y images are shown. Bars, 10 µm. (B, right) Percentage of p21-positive cells in cells expressing CagA or EGFP. Error bars indicate mean ± SD. n = 3. ***, P > 0.05. (C, left) Confocal x-y images of polarized MDCK cells transfected with an expression vector encoding HA-tagged CagA (WT, PR, and ΔCM/MKI, CagA lacking the PAR1/MARK-binding sequence) were stained with anti-HA antibody (green), anti-BrdU antibody (red), and DAPI (blue). An expression vector for enhanced GFP (EGFP) was used as a control. Bar, 10 µm. (C, right) Percentage of BrdU-positive cells in cells expressing CagA or EGFP. Error bars indicate mean ± SD. n = 5. **, P
Figure Legend Snippet: Induction of forced mitogenesis upon expression of CagA in polarized epithelial cells. (A) After expression of HA-tagged CagA in polarized MDCK cells, CagA-positive cells were visualized with anti-HA antibody (green). Polarized MDCK cells without CagA expression were used as a control. Nuclei in confocal x-z images were visualized by 4,6-diamidino-2-phenylindole (DAPI) staining (blue). Bars, 10 µm. (B, left) Polarized MDCK cells transfected with an HA-tagged CagA vector were stained with anti-HA antibody (green), anti-p21 antibody (red), and DAPI (blue). Confocal x-y images are shown. Bars, 10 µm. (B, right) Percentage of p21-positive cells in cells expressing CagA or EGFP. Error bars indicate mean ± SD. n = 3. ***, P > 0.05. (C, left) Confocal x-y images of polarized MDCK cells transfected with an expression vector encoding HA-tagged CagA (WT, PR, and ΔCM/MKI, CagA lacking the PAR1/MARK-binding sequence) were stained with anti-HA antibody (green), anti-BrdU antibody (red), and DAPI (blue). An expression vector for enhanced GFP (EGFP) was used as a control. Bar, 10 µm. (C, right) Percentage of BrdU-positive cells in cells expressing CagA or EGFP. Error bars indicate mean ± SD. n = 5. **, P

Techniques Used: Expressing, Staining, Transfection, Plasmid Preparation, Binding Assay, Sequencing

CagA expressed in polarized epithelial cells inhibits p21 accumulation via RhoA. (A, top left) Polarized MDCK cells were transfected with an expression vector for HA-tagged CagA in the presence or absence of C3 transferase, an inhibitor of RhoA. Cell lysates were immunoblotted with the indicated antibodies. (A, top right) Percentage of p21-positive cells in cells expressing CagA in the presence or absence of C3. Error bars indicate mean ± SD. n = 3. **, P
Figure Legend Snippet: CagA expressed in polarized epithelial cells inhibits p21 accumulation via RhoA. (A, top left) Polarized MDCK cells were transfected with an expression vector for HA-tagged CagA in the presence or absence of C3 transferase, an inhibitor of RhoA. Cell lysates were immunoblotted with the indicated antibodies. (A, top right) Percentage of p21-positive cells in cells expressing CagA in the presence or absence of C3. Error bars indicate mean ± SD. n = 3. **, P

Techniques Used: Transfection, Expressing, Plasmid Preparation

Growth inhibition of nonpolarized epithelial cells by CagA. (A) MKN28-derived WT-A10 cells that inducibly express HA-tagged CagA by tet-off system were cultured in the presence or absence of 0.2 µg/ml doxycycline (Dox). Cell lysates were subjected to immunoblotting with the indicated antibodies. (B) WT-A10 cells were cultured in the presence or absence of Dox. Cells were stained with propidium iodide and were subjected to cell cycle analysis using flow cytometry. Percentages of cells in G1 and S/G2/M phases are shown. Results were reproducible in three independent experiments. (C, left) WT-A10 cells and WT-A10–derived p21sh-6-12 cells, in which p21 was knocked down by specific siRNA, were induced to express CagA by depleting Dox from the culture for 24 h. Cell lysates were subjected to immunoblotting with anti-HA, anti-p21, and anti-actin antibodies. (C, right) Cells were stained with propidium iodide and were subjected to flow cytometric analysis. Percentages of cells in G1 and S/G2/M phases are shown. (D) WT-A10 cells expressing CagA were treated with 25 µM MEK inhibitor U0126 or control U0124. Cell lysates were subjected to immunoblotting. (E) WT-A10 cells were cultured for 5 d in the presence or absence of Dox to induce CagA expression, and senescence-associated β-galactosidase activity was visualized by staining cells with X-Gal (blue). Light micrograph images are shown. Bars, 100 µm. Representative gel (A, C, and D) and staining (E) images obtained from three independent experiments are shown.
Figure Legend Snippet: Growth inhibition of nonpolarized epithelial cells by CagA. (A) MKN28-derived WT-A10 cells that inducibly express HA-tagged CagA by tet-off system were cultured in the presence or absence of 0.2 µg/ml doxycycline (Dox). Cell lysates were subjected to immunoblotting with the indicated antibodies. (B) WT-A10 cells were cultured in the presence or absence of Dox. Cells were stained with propidium iodide and were subjected to cell cycle analysis using flow cytometry. Percentages of cells in G1 and S/G2/M phases are shown. Results were reproducible in three independent experiments. (C, left) WT-A10 cells and WT-A10–derived p21sh-6-12 cells, in which p21 was knocked down by specific siRNA, were induced to express CagA by depleting Dox from the culture for 24 h. Cell lysates were subjected to immunoblotting with anti-HA, anti-p21, and anti-actin antibodies. (C, right) Cells were stained with propidium iodide and were subjected to flow cytometric analysis. Percentages of cells in G1 and S/G2/M phases are shown. (D) WT-A10 cells expressing CagA were treated with 25 µM MEK inhibitor U0126 or control U0124. Cell lysates were subjected to immunoblotting. (E) WT-A10 cells were cultured for 5 d in the presence or absence of Dox to induce CagA expression, and senescence-associated β-galactosidase activity was visualized by staining cells with X-Gal (blue). Light micrograph images are shown. Bars, 100 µm. Representative gel (A, C, and D) and staining (E) images obtained from three independent experiments are shown.

Techniques Used: Inhibition, Derivative Assay, Cell Culture, Staining, Cell Cycle Assay, Flow Cytometry, Cytometry, Expressing, Activity Assay

Requirement of GEF-H1 for the inhibition of CagA-mediated p21 accumulation in polarized epithelial cells. (A, left) Polarized MDCK cells treated with 100 pmol GEF-H1 siRNA-1 or firefly luciferase–specific siRNA (control siRNA) were transfected with CagA or EGFP expression vector. Cells were stained with anti-p21 antibody (red, top, confocal x-y view) or anti–ZO-1 antibody (bottom, confocal x-z view). Bars, 10 µm. (A, right) Cell lysates were subjected to immunoblotting (top). Percentage of p21-positive cells in GEF-H1 knockdown MDCK cells expressing CagA or EGFP. Error bars indicate mean ± SD. n = 3. **, P
Figure Legend Snippet: Requirement of GEF-H1 for the inhibition of CagA-mediated p21 accumulation in polarized epithelial cells. (A, left) Polarized MDCK cells treated with 100 pmol GEF-H1 siRNA-1 or firefly luciferase–specific siRNA (control siRNA) were transfected with CagA or EGFP expression vector. Cells were stained with anti-p21 antibody (red, top, confocal x-y view) or anti–ZO-1 antibody (bottom, confocal x-z view). Bars, 10 µm. (A, right) Cell lysates were subjected to immunoblotting (top). Percentage of p21-positive cells in GEF-H1 knockdown MDCK cells expressing CagA or EGFP. Error bars indicate mean ± SD. n = 3. **, P

Techniques Used: Inhibition, Luciferase, Transfection, Expressing, Plasmid Preparation, Staining

Related Articles

Western Blot:

Article Title: Biochemical and functional characterization of Helicobacter pylori vesicles
Article Snippet: .. Samples were subsequently resuspended in SDS-PAGE sample buffer, separated by SDS-PAGE, and proteins detected in immunoblots using anti-CagA, anti-VacA and anti-BabA antibodies. ..

other:

Article Title: Regulation of p53 tumor suppressor by Helicobacter pylori in gastric epithelial cells
Article Snippet: Antibodies to the following proteins were used: p53(DO-1), p53(DO-7), p21(Ab-1), HDM2(Ab-1), and p73(Ab-3) from Calbiochem; anti-CagA from Austral Biologicals (San Ramon, CA); pAKT(Ser473), pHDM2(Ser166) and AKT from Cell Signaling; anti-GFP from Clontech; p53(CM-1) and p53(NCL-p53-505) from Novocastra (UK); anti-ubiquitin from Santa Cruz; AKT(pT308) from Epitomics (Burlingame, CA), and MDM2 (154–167) from Spring Bioscience (Pleasanton, CA).

SDS Page:

Article Title: Biochemical and functional characterization of Helicobacter pylori vesicles
Article Snippet: .. Samples were subsequently resuspended in SDS-PAGE sample buffer, separated by SDS-PAGE, and proteins detected in immunoblots using anti-CagA, anti-VacA and anti-BabA antibodies. ..

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    Austral Biologicals anti caga
    <t>CagA</t> is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and <t>VacA</t> respectively.
    Anti Caga, supplied by Austral Biologicals, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Austral Biologicals mouse monoclonal α caga antibody
    Role of EPIYA motifs in <t>CagA</t> phosphorylation during H. pylori infection was investigated with seven different α-phosphotyrosine antibodies. AGS cells were infected for 6-expressing H. pylori strains as indicated. The samples in Figure 4 were harvested after photographing. Phosphorylation of CagA was examined using the indicated α–phosphotyrosine antibodies. Loading of equal amounts of CagA from each sample was confirmed by probing with a monoclonal <t>α-CagA</t> antibody. A larger section of the ∼120−180 kDa range is shown and contains the phospho-CagA bands of different sizes (arrows) as well as a set of tyrosine-phosphorylated host cell proteins (red asterisks). The blue asterisk indicates a putative N-terminal fragment of CagA which sometimes appears on SDS-PAGE gels [23] .
    Mouse Monoclonal α Caga Antibody, supplied by Austral Biologicals, 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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    CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.

    Journal: Molecular Microbiology

    Article Title: Biochemical and functional characterization of Helicobacter pylori vesicles

    doi: 10.1111/j.1365-2958.2010.07307.x

    Figure Lengend Snippet: CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein. A. Electron micrographs of H. pylori strain P12. B. Electron micrographs of H. pylori strain P12Δ cagA . Bar length in A and B represents 0.5 µm. C. An enlargement of the indicated area in (A). D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.

    Article Snippet: Primary antibodies included: anti-BabA (AK277) ( ); anti-SabA (AK278) ( ); anti-CagA (Austral Biologicals); anti-VacA (AK204) ( ); anti-AlpB (AK262) ( ); anti-OipA (AK282) ( ); anti-ComB10 (AK252) ( ); anti-CagT; anti-CagX; anti-CagY; anti-CagM; anti-CagN; anti-VirD4.

    Techniques:

    Identification of major protein components in H. pylori vesicles. A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane. B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified. C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis. D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots. E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left. F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.

    Journal: Molecular Microbiology

    Article Title: Biochemical and functional characterization of Helicobacter pylori vesicles

    doi: 10.1111/j.1365-2958.2010.07307.x

    Figure Lengend Snippet: Identification of major protein components in H. pylori vesicles. A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane. B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified. C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis. D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots. E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left. F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.

    Article Snippet: Primary antibodies included: anti-BabA (AK277) ( ); anti-SabA (AK278) ( ); anti-CagA (Austral Biologicals); anti-VacA (AK204) ( ); anti-AlpB (AK262) ( ); anti-OipA (AK282) ( ); anti-ComB10 (AK252) ( ); anti-CagT; anti-CagX; anti-CagY; anti-CagM; anti-CagN; anti-VirD4.

    Techniques: SDS Page, Mass Spectrometry, Functional Assay, Expressing, Western Blot

    CagA regulates p53 levels in H. pylori -infected cells (A) AGS cells were cultured in the presence of the wild-type H. pylori strain 7.13 or isogenic cagA− or cagE− null mutants, and protein levels of p53 were assessed by Western blotting. (B) Left panel: gastric tissues harvested from gerbils infected with H. pylori strain 7.13 or isogenic cagA- null mutant at indicated time points were immunostained for p53 and quantitated using a blind protocol. Results are expressed as the percentage of p53 positive cells per sample. Mean values ( ) for cagA − and cagA + infected animals are shown. A dashed line depicts the average levels of p53 in the uninfected control animals. Right panel: representative immunohistochemical staining for p53 (x20) is shown for uninfected animals (1) and those infected with wild-type (2) or cagA − (3) isogenic H. pylori strains for 6 hours (at the peak of p53 increase). Levels of p53 were also analyzed by Western blotting with p53-specific antibody at 6 hours. (C) Analysis of HDM2 phosphorylation in AGS cells co-cultured with H. pylori strain J166 or its isogenic cagA− or cagE− derivatives. (D) Analysis of p53 ubiquitination in AGS cells co-cultured with the indicated isogenic H. pylori strains for 24 hours. Proteasomal degradation was inhibited with MG-132.

    Journal: Gastroenterology

    Article Title: Regulation of p53 tumor suppressor by Helicobacter pylori in gastric epithelial cells

    doi: 10.1053/j.gastro.2010.06.018

    Figure Lengend Snippet: CagA regulates p53 levels in H. pylori -infected cells (A) AGS cells were cultured in the presence of the wild-type H. pylori strain 7.13 or isogenic cagA− or cagE− null mutants, and protein levels of p53 were assessed by Western blotting. (B) Left panel: gastric tissues harvested from gerbils infected with H. pylori strain 7.13 or isogenic cagA- null mutant at indicated time points were immunostained for p53 and quantitated using a blind protocol. Results are expressed as the percentage of p53 positive cells per sample. Mean values ( ) for cagA − and cagA + infected animals are shown. A dashed line depicts the average levels of p53 in the uninfected control animals. Right panel: representative immunohistochemical staining for p53 (x20) is shown for uninfected animals (1) and those infected with wild-type (2) or cagA − (3) isogenic H. pylori strains for 6 hours (at the peak of p53 increase). Levels of p53 were also analyzed by Western blotting with p53-specific antibody at 6 hours. (C) Analysis of HDM2 phosphorylation in AGS cells co-cultured with H. pylori strain J166 or its isogenic cagA− or cagE− derivatives. (D) Analysis of p53 ubiquitination in AGS cells co-cultured with the indicated isogenic H. pylori strains for 24 hours. Proteasomal degradation was inhibited with MG-132.

    Article Snippet: Antibodies to the following proteins were used: p53(DO-1), p53(DO-7), p21(Ab-1), HDM2(Ab-1), and p73(Ab-3) from Calbiochem; anti-CagA from Austral Biologicals (San Ramon, CA); pAKT(Ser473), pHDM2(Ser166) and AKT from Cell Signaling; anti-GFP from Clontech; p53(CM-1) and p53(NCL-p53-505) from Novocastra (UK); anti-ubiquitin from Santa Cruz; AKT(pT308) from Epitomics (Burlingame, CA), and MDM2 (154–167) from Spring Bioscience (Pleasanton, CA).

    Techniques: Infection, Cell Culture, Western Blot, Mutagenesis, Immunohistochemistry, Staining

    CagA induces degradation of p53 (A) Left panel: p53-null Kato III cells were co-transfected with the indicated plasmids and GFP for 48 hours and then analyzed for p53 expression. Gel loading was normalized to GFP expression. HDM2 and p53 co-transfection was used as an additional positive control. Right panel: the same as the left panel but another p53-null osteosarcoma cell line, SaOs2, was used. (B) AGS cells were transfected with CagA-IRES-GFP (CagA) or empty IRES-GFP (Control) vectors. Twenty-four hours post-transfection cells were analyzed by immunofluorescence for p53 (red) in GFP-expressing cells (green). Nuclear p53 protein disappeared in 58% of CagA-expressing cells whereas only 13.5% of control GFP-positive cells were negative for p53. (C) Left panel: AGS cells that express CagA under control of tetracycline-inducible promoter were treated with hydrogen peroxide or left untreated and then analyzed for p53. Right panel: Control AGS cells (−DOX) or ones expressing CagA (+DOX) were treated with indicated concentrations of H 2 O 2 for 24 hours. Cell death was assessed by flow cytometry after propidium iodide staining. The proportion of cells in subG1 is shown. CagA significantly increased survival of cells treated with H 2 O 2 . **, p

    Journal: Gastroenterology

    Article Title: Regulation of p53 tumor suppressor by Helicobacter pylori in gastric epithelial cells

    doi: 10.1053/j.gastro.2010.06.018

    Figure Lengend Snippet: CagA induces degradation of p53 (A) Left panel: p53-null Kato III cells were co-transfected with the indicated plasmids and GFP for 48 hours and then analyzed for p53 expression. Gel loading was normalized to GFP expression. HDM2 and p53 co-transfection was used as an additional positive control. Right panel: the same as the left panel but another p53-null osteosarcoma cell line, SaOs2, was used. (B) AGS cells were transfected with CagA-IRES-GFP (CagA) or empty IRES-GFP (Control) vectors. Twenty-four hours post-transfection cells were analyzed by immunofluorescence for p53 (red) in GFP-expressing cells (green). Nuclear p53 protein disappeared in 58% of CagA-expressing cells whereas only 13.5% of control GFP-positive cells were negative for p53. (C) Left panel: AGS cells that express CagA under control of tetracycline-inducible promoter were treated with hydrogen peroxide or left untreated and then analyzed for p53. Right panel: Control AGS cells (−DOX) or ones expressing CagA (+DOX) were treated with indicated concentrations of H 2 O 2 for 24 hours. Cell death was assessed by flow cytometry after propidium iodide staining. The proportion of cells in subG1 is shown. CagA significantly increased survival of cells treated with H 2 O 2 . **, p

    Article Snippet: Antibodies to the following proteins were used: p53(DO-1), p53(DO-7), p21(Ab-1), HDM2(Ab-1), and p73(Ab-3) from Calbiochem; anti-CagA from Austral Biologicals (San Ramon, CA); pAKT(Ser473), pHDM2(Ser166) and AKT from Cell Signaling; anti-GFP from Clontech; p53(CM-1) and p53(NCL-p53-505) from Novocastra (UK); anti-ubiquitin from Santa Cruz; AKT(pT308) from Epitomics (Burlingame, CA), and MDM2 (154–167) from Spring Bioscience (Pleasanton, CA).

    Techniques: Transfection, Expressing, Cotransfection, Positive Control, Immunofluorescence, Flow Cytometry, Cytometry, Staining

    PC activation induced by VacA and CagA on THP-1 cells. Both VacA and CagA significantly ( P

    Journal: Infection and Immunity

    Article Title: Role of Activated Protein C in Helicobacter pylori-Associated Gastritis

    doi:

    Figure Lengend Snippet: PC activation induced by VacA and CagA on THP-1 cells. Both VacA and CagA significantly ( P

    Article Snippet: Recombinant VacA toxin, recombinant CagA from H. pylori , and polyclonal anti-VacA and anti-CagA antibodies were purchased from Austral Biologicals (San Ramon, Calif.).

    Techniques: Activation Assay

    Role of EPIYA motifs in CagA phosphorylation during H. pylori infection was investigated with seven different α-phosphotyrosine antibodies. AGS cells were infected for 6-expressing H. pylori strains as indicated. The samples in Figure 4 were harvested after photographing. Phosphorylation of CagA was examined using the indicated α–phosphotyrosine antibodies. Loading of equal amounts of CagA from each sample was confirmed by probing with a monoclonal α-CagA antibody. A larger section of the ∼120−180 kDa range is shown and contains the phospho-CagA bands of different sizes (arrows) as well as a set of tyrosine-phosphorylated host cell proteins (red asterisks). The blue asterisk indicates a putative N-terminal fragment of CagA which sometimes appears on SDS-PAGE gels [23] .

    Journal: PLoS ONE

    Article Title: Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

    doi: 10.1371/journal.pone.0096488

    Figure Lengend Snippet: Role of EPIYA motifs in CagA phosphorylation during H. pylori infection was investigated with seven different α-phosphotyrosine antibodies. AGS cells were infected for 6-expressing H. pylori strains as indicated. The samples in Figure 4 were harvested after photographing. Phosphorylation of CagA was examined using the indicated α–phosphotyrosine antibodies. Loading of equal amounts of CagA from each sample was confirmed by probing with a monoclonal α-CagA antibody. A larger section of the ∼120−180 kDa range is shown and contains the phospho-CagA bands of different sizes (arrows) as well as a set of tyrosine-phosphorylated host cell proteins (red asterisks). The blue asterisk indicates a putative N-terminal fragment of CagA which sometimes appears on SDS-PAGE gels [23] .

    Article Snippet: Membranes were incubated with the seven α-phosphotyrosine antibodies ( ) or mouse monoclonal α-CagA antibody (Austral Biologicals, San Ramon, CA, USA) according to the instructions of the manufacturer.

    Techniques: Infection, Expressing, SDS Page