Structured Review

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 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096488

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] .
Figure Legend 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] .

Techniques Used: Infection, Expressing, SDS Page

2) Product Images from "Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats"

Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056291

CagA EPIYA-C phosphorylation-functional and -defective H. pylori mutant strains. (A) Deduced CagA amino acid sequences, following nucleotide sequencing, depicting the EPIYA and EPIFA mutant motifs, as well as the MARK2-kinase inhibitor (CM) (shaded region). (B) Schematic representation of CagA protein expressed by the corresponding mutants. (C) Determination of CagA tyrosine phosphorylation by western blot utilizing α-CagA-pY972 antibody, which recognizes phosphorylated EPIYA-C motifs. CagA and GAPDH expression is also depicted, for control purposes.
Figure Legend Snippet: CagA EPIYA-C phosphorylation-functional and -defective H. pylori mutant strains. (A) Deduced CagA amino acid sequences, following nucleotide sequencing, depicting the EPIYA and EPIFA mutant motifs, as well as the MARK2-kinase inhibitor (CM) (shaded region). (B) Schematic representation of CagA protein expressed by the corresponding mutants. (C) Determination of CagA tyrosine phosphorylation by western blot utilizing α-CagA-pY972 antibody, which recognizes phosphorylated EPIYA-C motifs. CagA and GAPDH expression is also depicted, for control purposes.

Techniques Used: Functional Assay, Mutagenesis, Sequencing, Western Blot, Expressing

Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.
Figure Legend Snippet: Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.

Techniques Used: Derivative Assay, Infection, Mutagenesis, Immunoprecipitation, Western Blot, Immunodetection, Variant Assay

3) Product Images from "Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats"

Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056291

Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.
Figure Legend Snippet: Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.

Techniques Used: Derivative Assay, Infection, Mutagenesis, Immunoprecipitation, Western Blot, Immunodetection, Variant Assay

4) Product Images from "Tyrosine Phosphorylation of CagA from Chinese Helicobacter pylori Isolates in AGS Gastric Epithelial Cells"

Article Title: Tyrosine Phosphorylation of CagA from Chinese Helicobacter pylori Isolates in AGS Gastric Epithelial Cells

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.43.2.786-790.2005

CagA with two variable-region TPMs is less phosphorylated than CagA with three variable-region TPMs. (A) AGS cells were cocultured with H. pylori strains possessing two (strain Z11) or three (strains Z4, Z7, and Z23) variable-region EPIYA motifs for 6 h at 37°C before the cells were lysed and the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antiphosphotyrosine monoclonal antibodies (lower panel). The blots were then stripped and reprobed with anti-CagA polyclonal antibodies (upper panel). (B) Densitometric analysis of the degree of CagA phosphorylation, expressed as a ratio of phospho-Tyr intensity to CagA protein intensity. The data shown are representative of those from three separate experiments.
Figure Legend Snippet: CagA with two variable-region TPMs is less phosphorylated than CagA with three variable-region TPMs. (A) AGS cells were cocultured with H. pylori strains possessing two (strain Z11) or three (strains Z4, Z7, and Z23) variable-region EPIYA motifs for 6 h at 37°C before the cells were lysed and the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antiphosphotyrosine monoclonal antibodies (lower panel). The blots were then stripped and reprobed with anti-CagA polyclonal antibodies (upper panel). (B) Densitometric analysis of the degree of CagA phosphorylation, expressed as a ratio of phospho-Tyr intensity to CagA protein intensity. The data shown are representative of those from three separate experiments.

Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot

5) Product Images from "A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions"

Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004621

Sequence comparison of the three TPM sites in CagA proteins from different clinical H. pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A : CagA proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85 subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases and sequence alignment was done using the ClustalW2 program ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Panel B : To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.
Figure Legend Snippet: Sequence comparison of the three TPM sites in CagA proteins from different clinical H. pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A : CagA proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85 subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases and sequence alignment was done using the ClustalW2 program ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Panel B : To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.

Techniques Used: Sequencing, Co-Culture Assay, Binding Assay, Incubation, Expressing

The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with PI3-kinase. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC Y > F , and EPIYT-B Y > F all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed that only CagA wt and EPIYT-AC Y > F can bind to PI3-kinase, but not the EPIYT-B Y > F mutant, suggesting that EPIYT-B is phosphorylated and necessary for the interaction.
Figure Legend Snippet: The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with PI3-kinase. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC Y > F , and EPIYT-B Y > F all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed that only CagA wt and EPIYT-AC Y > F can bind to PI3-kinase, but not the EPIYT-B Y > F mutant, suggesting that EPIYT-B is phosphorylated and necessary for the interaction.

Techniques Used: Mutagenesis, Cell Culture, Expressing, Immunoprecipitation, Western Blot

PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and triple mutants were named as indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies (arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase. Panel C : After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase (p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different affinity to the PI3-kinase protein in AGS cells.
Figure Legend Snippet: PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and triple mutants were named as indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies (arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase. Panel C : After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase (p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different affinity to the PI3-kinase protein in AGS cells.

Techniques Used: Co-Culture Assay, Mutagenesis, Cell Culture, Expressing, Immunoprecipitation, SDS Page, Western Blot

6) Product Images from "Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats"

Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056291

Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.
Figure Legend Snippet: Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.

Techniques Used: Derivative Assay, Infection, Mutagenesis, Immunoprecipitation, Western Blot, Immunodetection, Variant Assay

7) Product Images from "Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096488

Variable recognition of synthetic 11-mer CagA phospho-peptides by seven commercial α-phosphotyrosine antibodies. ( A ) Dotblot analysis of the indicated phospho- and non-phospho peptides derived from single EPIYA-motifs A, B and C. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. ( B ) Quantification of spot intensities on Dotblots. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. Quantitation results are shown for three independent experiments.
Figure Legend Snippet: Variable recognition of synthetic 11-mer CagA phospho-peptides by seven commercial α-phosphotyrosine antibodies. ( A ) Dotblot analysis of the indicated phospho- and non-phospho peptides derived from single EPIYA-motifs A, B and C. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. ( B ) Quantification of spot intensities on Dotblots. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. Quantitation results are shown for three independent experiments.

Techniques Used: Derivative Assay, Quantitation 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] .
Figure Legend 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] .

Techniques Used: Infection, Expressing, SDS Page

Short EPIYA-phosphopeptides of H. pylori CagA are sufficient for detection by α-phosphotyrosine antibodies. ( A ) Typical Western CagA proteins of H. pylori such as that of strain 26695 [76] contain the EPIYA-A, EPIYA-B, and EPIYA-C segments as indicated. These motifs represent tyrosine phosphorylation sites, which can be phosphorylated by c-Abl and c-Src host kinases. ( B ) The indicated phospho- and non-phospho peptides of the EPIYA-A motif were synthesized and immobilized on PVDF membranes using a Dotblot apparatus. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. Quantified spot intensities of the Dotblots from three independent experiments are shown to the right. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. The results show that 11-mer and 9-mer phosphopeptides are sufficient for strong recognition by the antibodies. ( C ) Control Dotblot analyses used products of in vitro kinase reactions of c-Abl with either bacterial lysates (from H. pylori wild-type strain 26695 and isogenic Δ cagA mutant) or a purified recombinant CagA C-terminal fragment. Phosphorylated CagA proteins can be also detected by this Dotblot method using seven phosphotyrosine antibodies ( Table 1 ), while the non-phosphorylated CagA forms cannot.
Figure Legend Snippet: Short EPIYA-phosphopeptides of H. pylori CagA are sufficient for detection by α-phosphotyrosine antibodies. ( A ) Typical Western CagA proteins of H. pylori such as that of strain 26695 [76] contain the EPIYA-A, EPIYA-B, and EPIYA-C segments as indicated. These motifs represent tyrosine phosphorylation sites, which can be phosphorylated by c-Abl and c-Src host kinases. ( B ) The indicated phospho- and non-phospho peptides of the EPIYA-A motif were synthesized and immobilized on PVDF membranes using a Dotblot apparatus. All Dotblots were probed with the indicated commercial phosphotyrosine antibodies and exposed as described in the Material Methods section. Quantified spot intensities of the Dotblots from three independent experiments are shown to the right. Signal intensities were measured densitometrically with the Lumi-Imager F1 and revealed the percentage of phosphorylation signal per sample. The strongest spot on every Dotblot was set at 100% for each of the different α-phosphotyrosine antibodies as indicated. The results show that 11-mer and 9-mer phosphopeptides are sufficient for strong recognition by the antibodies. ( C ) Control Dotblot analyses used products of in vitro kinase reactions of c-Abl with either bacterial lysates (from H. pylori wild-type strain 26695 and isogenic Δ cagA mutant) or a purified recombinant CagA C-terminal fragment. Phosphorylated CagA proteins can be also detected by this Dotblot method using seven phosphotyrosine antibodies ( Table 1 ), while the non-phosphorylated CagA forms cannot.

Techniques Used: Western Blot, Synthesized, In Vitro, Mutagenesis, Purification, Recombinant

8) Product Images from "CagA and VacA Polymorphisms Do Not Correlate with Severity of Histopathological Lesions in Helicobacter pylori-Infected Greek Children ▿-Infected Greek Children ▿ †"

Article Title: CagA and VacA Polymorphisms Do Not Correlate with Severity of Histopathological Lesions in Helicobacter pylori-Infected Greek Children ▿-Infected Greek Children ▿ †

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00159-09

Phosphorylation and expression of CagA protein following infection of gastric epithelial cells with representative H. pylori clinical isolates. CagA tyrosine phosphorylation (A) was evaluated by immunoblotting (IB) with anti-CagA rabbit polyclonal antibody
Figure Legend Snippet: Phosphorylation and expression of CagA protein following infection of gastric epithelial cells with representative H. pylori clinical isolates. CagA tyrosine phosphorylation (A) was evaluated by immunoblotting (IB) with anti-CagA rabbit polyclonal antibody

Techniques Used: Expressing, Infection

9) Product Images from "c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains"

Article Title: c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI61143

Generation of phosphomimetic CagA mutants and their interaction with host signaling factors during H. pylori infection. ( A ) Site-directed Y > D mutagenesis of CagA EPIYA-A, EPIYA-B, and EPIYA-C to generate phosphomimetic mutants. Nontargeted tyrosines in adjacent EPIYA motifs were replaced by phenylalanines to avoid additional phosphorylation events per molecule. The resulting single, double, and triple mutants were named as indicated. ( B ) AGS cells were infected for 4 hours with CagA-expressing H. pylori strains as indicated. CagA phosphorylation was examined using α–PY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, but only H. pylori expressing WT CagA revealed a phosphorylation signal. The asterisk in the lower panel indicates antibody cross-reactivity with an unknown phosphorylated host cell protein. ( C – E ) AGS cells were infected for 4 hours with CagA-expressing H. pylori strains as indicated, and cell lysates were subjected to IP with α-CagA antibodies. Western blotting using α-Csk ( C ), α-PI3K ( D ), or α–SHP-2 ( E ) indicated that each of these factors formed a complex with the respective phosphomimetic CagA EPIYA Y > D mutant, but not with the nonphosphorylatable triple EPIYA-ABC Y > F mutant as control.
Figure Legend Snippet: Generation of phosphomimetic CagA mutants and their interaction with host signaling factors during H. pylori infection. ( A ) Site-directed Y > D mutagenesis of CagA EPIYA-A, EPIYA-B, and EPIYA-C to generate phosphomimetic mutants. Nontargeted tyrosines in adjacent EPIYA motifs were replaced by phenylalanines to avoid additional phosphorylation events per molecule. The resulting single, double, and triple mutants were named as indicated. ( B ) AGS cells were infected for 4 hours with CagA-expressing H. pylori strains as indicated. CagA phosphorylation was examined using α–PY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, but only H. pylori expressing WT CagA revealed a phosphorylation signal. The asterisk in the lower panel indicates antibody cross-reactivity with an unknown phosphorylated host cell protein. ( C – E ) AGS cells were infected for 4 hours with CagA-expressing H. pylori strains as indicated, and cell lysates were subjected to IP with α-CagA antibodies. Western blotting using α-Csk ( C ), α-PI3K ( D ), or α–SHP-2 ( E ) indicated that each of these factors formed a complex with the respective phosphomimetic CagA EPIYA Y > D mutant, but not with the nonphosphorylatable triple EPIYA-ABC Y > F mutant as control.

Techniques Used: Infection, Mutagenesis, Expressing, Western Blot

Dual infection of H. pylori strains expressing different single phosphorylatable or phosphomimetic EPIYA motifs induces AGS cell elongation. AGS cells were infected for 4 hours with CagA EPIYA Y > F ( A ) or EPIYA Y > D ( B ) mutant strains as indicated. The available single phosphorylatable or phosphomimetic EPIYA motifs for each double infection are indicated. The number of elongated cells in each experiment was quantitated in triplicate in 10 different 0.25-mm 2 fields, and CagA phosphorylation was examined using α–PY-99 and α-CagA antibodies (arrows). ( C ) AGS infection for 4 hours with the indicated CagA-expressing strains in the presence or absence of c-Src inhibitor PP2 (10 μM) or c-Abl inhibitor SKI-DV2-43 (1 μM) revealed significant changes in AGS cell elongation as quantitated in triplicate in 10 different 0.25-mm 2 fields. * P ≤ 0.01; ** P ≤ 0.001.
Figure Legend Snippet: Dual infection of H. pylori strains expressing different single phosphorylatable or phosphomimetic EPIYA motifs induces AGS cell elongation. AGS cells were infected for 4 hours with CagA EPIYA Y > F ( A ) or EPIYA Y > D ( B ) mutant strains as indicated. The available single phosphorylatable or phosphomimetic EPIYA motifs for each double infection are indicated. The number of elongated cells in each experiment was quantitated in triplicate in 10 different 0.25-mm 2 fields, and CagA phosphorylation was examined using α–PY-99 and α-CagA antibodies (arrows). ( C ) AGS infection for 4 hours with the indicated CagA-expressing strains in the presence or absence of c-Src inhibitor PP2 (10 μM) or c-Abl inhibitor SKI-DV2-43 (1 μM) revealed significant changes in AGS cell elongation as quantitated in triplicate in 10 different 0.25-mm 2 fields. * P ≤ 0.01; ** P ≤ 0.001.

Techniques Used: Infection, Expressing, Mutagenesis

In vitro phosphorylation of CagA mutants by c-Abl or c-Src kinases. ( A ) Site-directed Y > F mutagenesis of CagA EPIYA-A, EPIYA-B, and EPIYA-C in strain 26695. Single, double, and triple mutants were named as indicated. ( B and C ) Lysates of H. pylori expressing the mutated CagA EPIYA motifs were subjected to in vitro phosphorylation assays using recombinant c-Src kinase ( B ) or c-Abl kinase ( C ). Immunoblotting using α–PY-99 and α-CagA antibodies (arrows) indicated that both c-Src and c-Abl phosphorylated CagA in a different fashion. ( D ) Schematic diagram of the data, showing that c-Src only phosphorylated Y-972 in EPIYA-C, while Abl can phosphorylate Y-899, Y-918, and Y-972 in EPIYA-A, EPIYA-B, and EPIYA-C, respectively.
Figure Legend Snippet: In vitro phosphorylation of CagA mutants by c-Abl or c-Src kinases. ( A ) Site-directed Y > F mutagenesis of CagA EPIYA-A, EPIYA-B, and EPIYA-C in strain 26695. Single, double, and triple mutants were named as indicated. ( B and C ) Lysates of H. pylori expressing the mutated CagA EPIYA motifs were subjected to in vitro phosphorylation assays using recombinant c-Src kinase ( B ) or c-Abl kinase ( C ). Immunoblotting using α–PY-99 and α-CagA antibodies (arrows) indicated that both c-Src and c-Abl phosphorylated CagA in a different fashion. ( D ) Schematic diagram of the data, showing that c-Src only phosphorylated Y-972 in EPIYA-C, while Abl can phosphorylate Y-899, Y-918, and Y-972 in EPIYA-A, EPIYA-B, and EPIYA-C, respectively.

Techniques Used: In Vitro, Mutagenesis, Expressing, Recombinant

Role of EPIYA motifs in CagA phosphorylation and AGS cell elongation during H. pylori infection. ( A ) AGS cells were infected for 4 hours with CagA-expressing H. pylori strains as indicated. Phosphorylation of CagA was examined using α–PY-99 and α-CagA antibodies (arrows). ( B ) The number of elongated cells in each experiment was quantitated in triplicate in 10 different 0.25-mm 2 fields. ( C ) Phase-contrast micrographs of AGS cells infected with the different strains as indicated. ** P ≤ 0.001.
Figure Legend Snippet: Role of EPIYA motifs in CagA phosphorylation and AGS cell elongation during H. pylori infection. ( A ) AGS cells were infected for 4 hours with CagA-expressing H. pylori strains as indicated. Phosphorylation of CagA was examined using α–PY-99 and α-CagA antibodies (arrows). ( B ) The number of elongated cells in each experiment was quantitated in triplicate in 10 different 0.25-mm 2 fields. ( C ) Phase-contrast micrographs of AGS cells infected with the different strains as indicated. ** P ≤ 0.001.

Techniques Used: Infection, Expressing

Analysis of CagA PY protein species during infection with H. pylori by 1-DE and 2-DE. ( A ). ( B ) AGS cells were infected for the indicated times with strain 26695. The resulting protein lysates were separated by 1-DE, and phosphorylation of injected CagA was examined using α–PY-99 and α-CagA antibodies (arrows). ( C ) Separation of CagA protein species from B by 2-DE. Depending on the time of infection, full-length CagA PY appeared as 1 spot (spot 1, red arrows, pI = 7.0) or 2 spots (spots 1 and 2; spot 2, green arrows, pI = 6.5) as indicated. The α-CagA antibody probe revealed a third spot (spot 3, blue arrows, pI = 7.5). Overlay of both exposures yielded 2 or 3 spots as shown. Strain TN2-GF4 exhibited the same pattern as 26695 (bottom). ( D ) Inhibition of Src with PP2 (10 μM) or Abl with SKI-DV2-43 (1 μM) revealed significant changes in spot intensity depending on the time of infection.
Figure Legend Snippet: Analysis of CagA PY protein species during infection with H. pylori by 1-DE and 2-DE. ( A ). ( B ) AGS cells were infected for the indicated times with strain 26695. The resulting protein lysates were separated by 1-DE, and phosphorylation of injected CagA was examined using α–PY-99 and α-CagA antibodies (arrows). ( C ) Separation of CagA protein species from B by 2-DE. Depending on the time of infection, full-length CagA PY appeared as 1 spot (spot 1, red arrows, pI = 7.0) or 2 spots (spots 1 and 2; spot 2, green arrows, pI = 6.5) as indicated. The α-CagA antibody probe revealed a third spot (spot 3, blue arrows, pI = 7.5). Overlay of both exposures yielded 2 or 3 spots as shown. Strain TN2-GF4 exhibited the same pattern as 26695 (bottom). ( D ) Inhibition of Src with PP2 (10 μM) or Abl with SKI-DV2-43 (1 μM) revealed significant changes in spot intensity depending on the time of infection.

Techniques Used: Infection, Injection, Inhibition

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Tyrosine Phosphorylation of CagA from Chinese Helicobacter pylori Isolates in AGS Gastric Epithelial Cells
Article Snippet: .. Samples were heated at 100°C for 5 min before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antiphosphotyrosine monoclonal antibodies (clone PY99; Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-CagA polyclonal antibodies (Austral Biologicals, San Ramon, Calif.). .. Quantification of the degree of CagA phosphorylation was performed by densitometry with a Bio-Rad (Hemel Hempstead, United Kingdom) GS-800 calibrated densitometer and Quantity One software and was expressed as a ratio of phospho-CagA to total CagA.

Variant Assay:

Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats
Article Snippet: .. We also observed that following α-TAK1 immunoprecipitation, the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) failed to detect the P12AB CagA variant ( ) possibly because it maps exactly on the EPIYA-C domains. ..

Blocking Assay:

Article Title: Exosomes as nanocarriers for systemic delivery of the Helicobacter pylori virulence factor CagA
Article Snippet: .. After blocking with 0.5% skim milk or Blocking One-P (Nacalai Tesque Inc., Kyoto, Japan) in TBST (20 mM Tris, 500 mM NaCl, pH 7.4, 0.05% Tween 20), membranes were blotted with the following primary antibodies: anti-HA antibodies (3F10; Roche), anti-CagA antibodies (AUSTRAL Biologicals), anti-phosphotyrosine antibodies (ab10321 [PY20]; Abcam), anti-CD9 antibodies (ab92726; Abcam), and anti-HSP70 antibodies (ab5439; Abcam). .. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and ECL Western Blotting Detection Reagents (GE Healthcare).

Immunoprecipitation:

Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats
Article Snippet: .. We also observed that following α-TAK1 immunoprecipitation, the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) failed to detect the P12AB CagA variant ( ) possibly because it maps exactly on the EPIYA-C domains. ..

Incubation:

Article Title: Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains
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. .. Phosphorylated and non-phosphorylated CagA proteins were detected using horseradish peroxidase–conjugated anti-mouse or anti-rabbit polyvalent sheep immunoglobulin secondary antibodies in the ECL Plus chemoluminescence Western blot system of GE Healthcare – .

Article Title: Induction of TLR-2 and TLR-5 Expression by Helicobacter pylori Switches cagPAI-Dependent Signalling Leading to the Secretion of IL-8 and TNF-?
Article Snippet: .. Phosphorylated and non-phosphorylated CagA proteins were detected by incubation of the membranes with a mouse monoclonal α-phosphotyrosine antibody PY99 (Santa Cruz, USA) and a rabbit polyclonal α-CagA antibody (Austral Biologicals, USA). .. Monoclonal antibody recognizing phosphorylated IκB at S-32 and rabbit polyclonal antibody recognizing phoshorylated IRAK-1 at S-376 were purchased from NEB cell signalling (USA).

Western Blot:

Article Title: Tyrosine Phosphorylation of CagA from Chinese Helicobacter pylori Isolates in AGS Gastric Epithelial Cells
Article Snippet: .. Samples were heated at 100°C for 5 min before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antiphosphotyrosine monoclonal antibodies (clone PY99; Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-CagA polyclonal antibodies (Austral Biologicals, San Ramon, Calif.). .. Quantification of the degree of CagA phosphorylation was performed by densitometry with a Bio-Rad (Hemel Hempstead, United Kingdom) GS-800 calibrated densitometer and Quantity One software and was expressed as a ratio of phospho-CagA to total CagA.

Recombinant:

Article Title: Role of Activated Protein C in Helicobacter pylori-Associated Gastritis
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.). .. Bovine serum albumin (BSA), RPMI 1640 medium, and recombinant hirudin and aprotinin (an inhibitor of APC) were from Sigma Chemical (St. Louis, Mo.), and the APC chromogenic substrate, S-2366, was from Chromogenix AB (Molndal, Sweden).

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    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 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal α caga antibody/product/Austral Biologicals
    Average 86 stars, based on 12 article reviews
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    85
    Austral Biologicals α caga monoclonal antibody
    <t>CagA</t> EPIYA-C phosphorylation-functional and -defective H. pylori mutant strains. (A) Deduced CagA amino acid sequences, following nucleotide sequencing, depicting the EPIYA and EPIFA mutant motifs, as well as the MARK2-kinase inhibitor (CM) (shaded region). (B) Schematic representation of CagA protein expressed by the corresponding mutants. (C) Determination of CagA tyrosine phosphorylation by western blot utilizing <t>α-CagA-pY972</t> antibody, which recognizes phosphorylated EPIYA-C motifs. CagA and GAPDH expression is also depicted, for control purposes.
    α Caga Monoclonal Antibody, supplied by Austral Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α caga monoclonal antibody/product/Austral Biologicals
    Average 85 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    α caga monoclonal antibody - by Bioz Stars, 2020-09
    85/100 stars
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    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

    Sequence comparison of the three TPM sites in CagA proteins from different clinical H. pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A : CagA proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85 subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases and sequence alignment was done using the ClustalW2 program ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Panel B : To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.

    Journal: PLoS Pathogens

    Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    doi: 10.1371/journal.ppat.1004621

    Figure Lengend Snippet: Sequence comparison of the three TPM sites in CagA proteins from different clinical H. pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A : CagA proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85 subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases and sequence alignment was done using the ClustalW2 program ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Panel B : To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.

    Article Snippet: Rabbit polyclonal and mouse monoclonal α-CagA antibodies were from Austral Biologicals, or from Emd Millipore Corporation (Billerica MA).

    Techniques: Sequencing, Co-Culture Assay, Binding Assay, Incubation, Expressing

    The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with PI3-kinase. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC Y > F , and EPIYT-B Y > F all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed that only CagA wt and EPIYT-AC Y > F can bind to PI3-kinase, but not the EPIYT-B Y > F mutant, suggesting that EPIYT-B is phosphorylated and necessary for the interaction.

    Journal: PLoS Pathogens

    Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    doi: 10.1371/journal.ppat.1004621

    Figure Lengend Snippet: The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with PI3-kinase. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC Y > F , and EPIYT-B Y > F all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed that only CagA wt and EPIYT-AC Y > F can bind to PI3-kinase, but not the EPIYT-B Y > F mutant, suggesting that EPIYT-B is phosphorylated and necessary for the interaction.

    Article Snippet: Rabbit polyclonal and mouse monoclonal α-CagA antibodies were from Austral Biologicals, or from Emd Millipore Corporation (Billerica MA).

    Techniques: Mutagenesis, Cell Culture, Expressing, Immunoprecipitation, Western Blot

    PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and triple mutants were named as indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies (arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase. Panel C : After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase (p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different affinity to the PI3-kinase protein in AGS cells.

    Journal: PLoS Pathogens

    Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    doi: 10.1371/journal.ppat.1004621

    Figure Lengend Snippet: PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and triple mutants were named as indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies (arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase. Panel C : After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase (p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different affinity to the PI3-kinase protein in AGS cells.

    Article Snippet: Rabbit polyclonal and mouse monoclonal α-CagA antibodies were from Austral Biologicals, or from Emd Millipore Corporation (Billerica MA).

    Techniques: Co-Culture Assay, Mutagenesis, Cell Culture, Expressing, Immunoprecipitation, SDS Page, Western Blot

    CagA EPIYA-C phosphorylation-functional and -defective H. pylori mutant strains. (A) Deduced CagA amino acid sequences, following nucleotide sequencing, depicting the EPIYA and EPIFA mutant motifs, as well as the MARK2-kinase inhibitor (CM) (shaded region). (B) Schematic representation of CagA protein expressed by the corresponding mutants. (C) Determination of CagA tyrosine phosphorylation by western blot utilizing α-CagA-pY972 antibody, which recognizes phosphorylated EPIYA-C motifs. CagA and GAPDH expression is also depicted, for control purposes.

    Journal: PLoS ONE

    Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats

    doi: 10.1371/journal.pone.0056291

    Figure Lengend Snippet: CagA EPIYA-C phosphorylation-functional and -defective H. pylori mutant strains. (A) Deduced CagA amino acid sequences, following nucleotide sequencing, depicting the EPIYA and EPIFA mutant motifs, as well as the MARK2-kinase inhibitor (CM) (shaded region). (B) Schematic representation of CagA protein expressed by the corresponding mutants. (C) Determination of CagA tyrosine phosphorylation by western blot utilizing α-CagA-pY972 antibody, which recognizes phosphorylated EPIYA-C motifs. CagA and GAPDH expression is also depicted, for control purposes.

    Article Snippet: We also observed that following α-TAK1 immunoprecipitation, the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) failed to detect the P12AB CagA variant ( ) possibly because it maps exactly on the EPIYA-C domains.

    Techniques: Functional Assay, Mutagenesis, Sequencing, Western Blot, Expressing

    Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.

    Journal: PLoS ONE

    Article Title: Presence of Terminal EPIYA Phosphorylation Motifs in Helicobacter pylori CagA Contributes to IL-8 Secretion, Irrespective of the Number of Repeats

    doi: 10.1371/journal.pone.0056291

    Figure Lengend Snippet: Immuno-detection of TAK1 and CagA in α-CagA or α-TAK1 respective immuno-precipitated lysates derived from AGS cells infected with H. pylori CagA mutant strains, at 1 hour post infection. (Panel A) Immunoprecipitation utilizing polyclonal α-CagA antibody (Austral Biologicals) and TAK1 western blot immunodetection utilizing monoclonal α-TAK1 antibody. Immunoprecipitation utilizing monoclonal α-TAK1 antibody and CagA immunodetection utilizing an α-CagA monoclonal antibody raised against the C-terminal (Panel B) or the N-terminal (Panel C) end of CagA protein. Note that the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) maps exactly on the EPIYA-C domains and fails to detect the P12AB CagA variant in panel B.

    Article Snippet: We also observed that following α-TAK1 immunoprecipitation, the α-CagA monoclonal antibody raised against the C-terminal (Austral Biologicals) failed to detect the P12AB CagA variant ( ) possibly because it maps exactly on the EPIYA-C domains.

    Techniques: Derivative Assay, Infection, Mutagenesis, Immunoprecipitation, Western Blot, Immunodetection, Variant Assay

    Sequence comparison of the three TPM sites in CagA proteins from different clinical H. pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A : CagA proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85 subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases and sequence alignment was done using the ClustalW2 program ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Panel B : To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.

    Journal: PLoS Pathogens

    Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    doi: 10.1371/journal.ppat.1004621

    Figure Lengend Snippet: Sequence comparison of the three TPM sites in CagA proteins from different clinical H. pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A : CagA proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85 subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases and sequence alignment was done using the ClustalW2 program ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Panel B : To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.

    Article Snippet: Two micrograms of the monoclonal α-CagA antibody (Austral Biologicals, San Ramon CA) or polyclonal antibody against the p85 subunit of PI3-kinase (Santa Cruz Biotechnology, Dallas TX) were added to the supernatant and incubated overnight at 4°C on a shaker.

    Techniques: Sequencing, Co-Culture Assay, Binding Assay, Incubation, Expressing

    The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with PI3-kinase. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC Y > F , and EPIYT-B Y > F all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed that only CagA wt and EPIYT-AC Y > F can bind to PI3-kinase, but not the EPIYT-B Y > F mutant, suggesting that EPIYT-B is phosphorylated and necessary for the interaction.

    Journal: PLoS Pathogens

    Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    doi: 10.1371/journal.ppat.1004621

    Figure Lengend Snippet: The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with PI3-kinase. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows). All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC Y > F , and EPIYT-B Y > F all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed that only CagA wt and EPIYT-AC Y > F can bind to PI3-kinase, but not the EPIYT-B Y > F mutant, suggesting that EPIYT-B is phosphorylated and necessary for the interaction.

    Article Snippet: Two micrograms of the monoclonal α-CagA antibody (Austral Biologicals, San Ramon CA) or polyclonal antibody against the p85 subunit of PI3-kinase (Santa Cruz Biotechnology, Dallas TX) were added to the supernatant and incubated overnight at 4°C on a shaker.

    Techniques: Mutagenesis, Cell Culture, Expressing, Immunoprecipitation, Western Blot

    PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and triple mutants were named as indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies (arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase. Panel C : After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase (p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different affinity to the PI3-kinase protein in AGS cells.

    Journal: PLoS Pathogens

    Article Title: A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    doi: 10.1371/journal.ppat.1004621

    Figure Lengend Snippet: PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A : Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single and triple mutants were named as indicated and complemented into the H. pylori Δ cagA mutant. Panel B : AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies (arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase. Panel C : After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase (p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different affinity to the PI3-kinase protein in AGS cells.

    Article Snippet: Two micrograms of the monoclonal α-CagA antibody (Austral Biologicals, San Ramon CA) or polyclonal antibody against the p85 subunit of PI3-kinase (Santa Cruz Biotechnology, Dallas TX) were added to the supernatant and incubated overnight at 4°C on a shaker.

    Techniques: Co-Culture Assay, Mutagenesis, Cell Culture, Expressing, Immunoprecipitation, SDS Page, Western Blot