Structured Review

Austral Biologicals rabbit polyclonal α caga antibody
<t>CagA</t> phosphorylation at EPIYA-motifs during H. pylori infection of AGS cells was investigated using seven different α-phosphotyrosine antibodies. Seven different CagA-expressing East Asian-type H. pylori strains as well as the T4SS-inactive negative control (Shi470 ΔcagL ) were used for infection studies on AGS cells. The infection was monitored over 6 h and the samples shown in Fig. 5 were harvested after photographing. Tyrosine phosphorylation of EPIYA-motifs in CagA of the seven different strains was analyzed with the indicated α-phosphotyrosine antibodies as previously described [ 48 ]. Presence of equal amounts of CagA from each sample was approved using a monoclonal <t>α-CagA</t> antibody. The ~120-170 kDa section of the gels is shown. Arrows indicate the phospho-CagA bands, while red asterisks mark bands of various tyrosine-phosphorylated host cell proteins
Rabbit Polyclonal α Caga Antibody, supplied by Austral Biologicals, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal α caga antibody/product/Austral Biologicals
Average 88 stars, based on 68 article reviews
Price from $9.99 to $1999.99
rabbit polyclonal α caga antibody - by Bioz Stars, 2020-09
88/100 stars

Images

1) Product Images from "Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains"

Article Title: Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains

Journal: BMC Microbiology

doi: 10.1186/s12866-016-0820-6

CagA phosphorylation at EPIYA-motifs during H. pylori infection of AGS cells was investigated using seven different α-phosphotyrosine antibodies. Seven different CagA-expressing East Asian-type H. pylori strains as well as the T4SS-inactive negative control (Shi470 ΔcagL ) were used for infection studies on AGS cells. The infection was monitored over 6 h and the samples shown in Fig. 5 were harvested after photographing. Tyrosine phosphorylation of EPIYA-motifs in CagA of the seven different strains was analyzed with the indicated α-phosphotyrosine antibodies as previously described [ 48 ]. Presence of equal amounts of CagA from each sample was approved using a monoclonal α-CagA antibody. The ~120-170 kDa section of the gels is shown. Arrows indicate the phospho-CagA bands, while red asterisks mark bands of various tyrosine-phosphorylated host cell proteins
Figure Legend Snippet: CagA phosphorylation at EPIYA-motifs during H. pylori infection of AGS cells was investigated using seven different α-phosphotyrosine antibodies. Seven different CagA-expressing East Asian-type H. pylori strains as well as the T4SS-inactive negative control (Shi470 ΔcagL ) were used for infection studies on AGS cells. The infection was monitored over 6 h and the samples shown in Fig. 5 were harvested after photographing. Tyrosine phosphorylation of EPIYA-motifs in CagA of the seven different strains was analyzed with the indicated α-phosphotyrosine antibodies as previously described [ 48 ]. Presence of equal amounts of CagA from each sample was approved using a monoclonal α-CagA antibody. The ~120-170 kDa section of the gels is shown. Arrows indicate the phospho-CagA bands, while red asterisks mark bands of various tyrosine-phosphorylated host cell proteins

Techniques Used: Infection, Expressing, Negative Control

2) 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

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.

Incubation:

Article Title: Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains
Article Snippet: .. After blocking the membranes in TBST buffer with 5 % skim milk or with 3 % bovine serum albumin (BSA) for 1 hour at room temperature, they were incubated with rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA) or with the seven commercial α-phosphotyrosine antibodies (Table ). ..

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: Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA
Article Snippet: .. CagA proteins were detected by incubation of the membranes with a rabbit polyclonal α‐CagA antibody (Austral Biologicals, USA). .. Monoclonal antibodies HecD1 and H‐108 against the extracellular domain of E‐cadherin were obtained from Calbiochem and Santa Cruz respectively.

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).

Blocking Assay:

Article Title: Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains
Article Snippet: .. After blocking the membranes in TBST buffer with 5 % skim milk or with 3 % bovine serum albumin (BSA) for 1 hour at room temperature, they were incubated with rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA) or with the seven commercial α-phosphotyrosine antibodies (Table ). ..

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.

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    Austral Biologicals rabbit polyclonal α caga antibody
    <t>CagA</t> injection by H. pylori cannot be achieved in infected HEK293 cell lines but in AGS gastric epithelial cells. Western blot analysis of (A) AGS, (B) HEK293-TLR2, (C) HEK293 and (D) HEK293-TLR5 cells infected with H. pylori wild-type strains P1, P12, P310 or 26695 for 6 hours. Phosphorylation of injected CagA was monitored using phosphotyrosine α-PY-99 and <t>α-CagA</t> antibodies. Red arrows indicate the position of phosphorylated CagA on the blot. Western blots for the house keeping gene GAPDH served as loading control.
    Rabbit Polyclonal α Caga Antibody, supplied by Austral Biologicals, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal α caga antibody/product/Austral Biologicals
    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal α caga antibody - by Bioz Stars, 2020-09
    88/100 stars
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    CagA injection by H. pylori cannot be achieved in infected HEK293 cell lines but in AGS gastric epithelial cells. Western blot analysis of (A) AGS, (B) HEK293-TLR2, (C) HEK293 and (D) HEK293-TLR5 cells infected with H. pylori wild-type strains P1, P12, P310 or 26695 for 6 hours. Phosphorylation of injected CagA was monitored using phosphotyrosine α-PY-99 and α-CagA antibodies. Red arrows indicate the position of phosphorylated CagA on the blot. Western blots for the house keeping gene GAPDH served as loading control.

    Journal: PLoS ONE

    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-?

    doi: 10.1371/journal.pone.0019614

    Figure Lengend Snippet: CagA injection by H. pylori cannot be achieved in infected HEK293 cell lines but in AGS gastric epithelial cells. Western blot analysis of (A) AGS, (B) HEK293-TLR2, (C) HEK293 and (D) HEK293-TLR5 cells infected with H. pylori wild-type strains P1, P12, P310 or 26695 for 6 hours. Phosphorylation of injected CagA was monitored using phosphotyrosine α-PY-99 and α-CagA antibodies. Red arrows indicate the position of phosphorylated CagA on the blot. Western blots for the house keeping gene GAPDH served as loading control.

    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).

    Techniques: Injection, Infection, Western Blot

    Elevated E-cadherin cleavage and CagA translocation in host cells by overexpression of HtrA. Polarized MKN-28 cells were infected with IPTG-induced or control Hp wt or Hp htrA 26695 for 24 h. a Cell pellets and supernatants were prepared and subjected to α-E-cadherin Western blotting. The full-length E-cadherin signals (130 kDa) in the cell pellets and cleaved-off NTF-domain (90 kDa) in the culture supernatants are marked with arrows . b Translocation and phosphorylation of CagA ( arrow ) were analyzed using α-PY-99 and α-CagA antibodies, respectively. The asterisk marks a phosphorylated 125 kDa host cell protein migrating below CagA. c The band intensities of cleaved E-cadherin NTF-fragments and phospho-CagA signals were quantified densitometrically. The relative amounts of NTF-fragment and phosphorylated CagA are given in “fold change”. All experiments were done in triplicates

    Journal: Gut Pathogens

    Article Title: Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori

    doi: 10.1186/s13099-017-0189-6

    Figure Lengend Snippet: Elevated E-cadherin cleavage and CagA translocation in host cells by overexpression of HtrA. Polarized MKN-28 cells were infected with IPTG-induced or control Hp wt or Hp htrA 26695 for 24 h. a Cell pellets and supernatants were prepared and subjected to α-E-cadherin Western blotting. The full-length E-cadherin signals (130 kDa) in the cell pellets and cleaved-off NTF-domain (90 kDa) in the culture supernatants are marked with arrows . b Translocation and phosphorylation of CagA ( arrow ) were analyzed using α-PY-99 and α-CagA antibodies, respectively. The asterisk marks a phosphorylated 125 kDa host cell protein migrating below CagA. c The band intensities of cleaved E-cadherin NTF-fragments and phospho-CagA signals were quantified densitometrically. The relative amounts of NTF-fragment and phosphorylated CagA are given in “fold change”. All experiments were done in triplicates

    Article Snippet: Antibodies The following antibodies were purchased: rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA), monoclonal pan-phosphotyrosine α-PY99 (Santa Cruz, Santa Cruz, CA/USA), rabbit α-GAPDH (Santa Cruz), rabbit α-H. pylori (Dako, Glostrup/Denmark) and two monoclonal antibodies directed against the extracellular domain of E-cadherin, H-108 (Santa Cruz) and CD324 (BD Biosciences, San Jose, CA/USA).

    Techniques: Translocation Assay, Over Expression, Infection, Western Blot

    Chromosomal introduction of a second, inducible htrA gene copy in H. pylori. a Schematic presentation of the IPTG-inducible htrA 26695 expression construct with the chloramphenicol acetyl-transferase (CAT) cassette ( top ), which was cloned into the plasticity region of H. pylori between genes HP0999 and HP1000 ( bottom ). b This construct was transformed into H. pylori wild-type ( Hp wt) strains 26695 and P12, and the expression of HtrA was investigated after 24 h growth in BHI medium in the presence or absence of 1 or 2 mM IPTG, respectively. The results for strain 26695 are shown. Western blotting using α-FlaA and α-CagA antibodies served as controls, indicating that equal amounts of proteins are present in the samples. c The band intensities of HtrA proteins were quantified densitometrically. The relative protein expression is given in “fold change”

    Journal: Gut Pathogens

    Article Title: Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori

    doi: 10.1186/s13099-017-0189-6

    Figure Lengend Snippet: Chromosomal introduction of a second, inducible htrA gene copy in H. pylori. a Schematic presentation of the IPTG-inducible htrA 26695 expression construct with the chloramphenicol acetyl-transferase (CAT) cassette ( top ), which was cloned into the plasticity region of H. pylori between genes HP0999 and HP1000 ( bottom ). b This construct was transformed into H. pylori wild-type ( Hp wt) strains 26695 and P12, and the expression of HtrA was investigated after 24 h growth in BHI medium in the presence or absence of 1 or 2 mM IPTG, respectively. The results for strain 26695 are shown. Western blotting using α-FlaA and α-CagA antibodies served as controls, indicating that equal amounts of proteins are present in the samples. c The band intensities of HtrA proteins were quantified densitometrically. The relative protein expression is given in “fold change”

    Article Snippet: Antibodies The following antibodies were purchased: rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA), monoclonal pan-phosphotyrosine α-PY99 (Santa Cruz, Santa Cruz, CA/USA), rabbit α-GAPDH (Santa Cruz), rabbit α-H. pylori (Dako, Glostrup/Denmark) and two monoclonal antibodies directed against the extracellular domain of E-cadherin, H-108 (Santa Cruz) and CD324 (BD Biosciences, San Jose, CA/USA).

    Techniques: Expressing, Construct, Chloramphenicol Acetyltransferase Assay, Clone Assay, Transformation Assay, Western Blot

    Elevated HtrA secretion levels in H. pylori does not affect the extracellular delivery of VacA and GGT. a H. pylori wild-type strain 26695 ( Hp wt) and 26695 transformed with htrA 26695 were grown for 24 h in BHI broth medium with 1% β-cyclodextrin in the presence or absence of 1 or 2 mM IPTG, respectively. Bacteria-free supernatants were prepared and subjected to Western blotting using α-HtrA, α-VacA and α-GGT antibodies. The blots against α-CagA served as control. The band intensities of secreted HtrA ( b ) as well as VacA and GGT ( c ) were quantified densitometrically. The relative protein expression is given in “fold change”. All secretion assays were done in triplicates

    Journal: Gut Pathogens

    Article Title: Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori

    doi: 10.1186/s13099-017-0189-6

    Figure Lengend Snippet: Elevated HtrA secretion levels in H. pylori does not affect the extracellular delivery of VacA and GGT. a H. pylori wild-type strain 26695 ( Hp wt) and 26695 transformed with htrA 26695 were grown for 24 h in BHI broth medium with 1% β-cyclodextrin in the presence or absence of 1 or 2 mM IPTG, respectively. Bacteria-free supernatants were prepared and subjected to Western blotting using α-HtrA, α-VacA and α-GGT antibodies. The blots against α-CagA served as control. The band intensities of secreted HtrA ( b ) as well as VacA and GGT ( c ) were quantified densitometrically. The relative protein expression is given in “fold change”. All secretion assays were done in triplicates

    Article Snippet: Antibodies The following antibodies were purchased: rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA), monoclonal pan-phosphotyrosine α-PY99 (Santa Cruz, Santa Cruz, CA/USA), rabbit α-GAPDH (Santa Cruz), rabbit α-H. pylori (Dako, Glostrup/Denmark) and two monoclonal antibodies directed against the extracellular domain of E-cadherin, H-108 (Santa Cruz) and CD324 (BD Biosciences, San Jose, CA/USA).

    Techniques: Transformation Assay, Western Blot, Expressing

    CagA phosphorylation at EPIYA-motifs during H. pylori infection of AGS cells was investigated using seven different α-phosphotyrosine antibodies. Seven different CagA-expressing East Asian-type H. pylori strains as well as the T4SS-inactive negative control (Shi470 ΔcagL ) were used for infection studies on AGS cells. The infection was monitored over 6 h and the samples shown in Fig. 5 were harvested after photographing. Tyrosine phosphorylation of EPIYA-motifs in CagA of the seven different strains was analyzed with the indicated α-phosphotyrosine antibodies as previously described [ 48 ]. Presence of equal amounts of CagA from each sample was approved using a monoclonal α-CagA antibody. The ~120-170 kDa section of the gels is shown. Arrows indicate the phospho-CagA bands, while red asterisks mark bands of various tyrosine-phosphorylated host cell proteins

    Journal: BMC Microbiology

    Article Title: Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains

    doi: 10.1186/s12866-016-0820-6

    Figure Lengend Snippet: CagA phosphorylation at EPIYA-motifs during H. pylori infection of AGS cells was investigated using seven different α-phosphotyrosine antibodies. Seven different CagA-expressing East Asian-type H. pylori strains as well as the T4SS-inactive negative control (Shi470 ΔcagL ) were used for infection studies on AGS cells. The infection was monitored over 6 h and the samples shown in Fig. 5 were harvested after photographing. Tyrosine phosphorylation of EPIYA-motifs in CagA of the seven different strains was analyzed with the indicated α-phosphotyrosine antibodies as previously described [ 48 ]. Presence of equal amounts of CagA from each sample was approved using a monoclonal α-CagA antibody. The ~120-170 kDa section of the gels is shown. Arrows indicate the phospho-CagA bands, while red asterisks mark bands of various tyrosine-phosphorylated host cell proteins

    Article Snippet: After blocking the membranes in TBST buffer with 5 % skim milk or with 3 % bovine serum albumin (BSA) for 1 hour at room temperature, they were incubated with rabbit polyclonal α-CagA antibody (Austral Biologicals, San Ramon, CA/USA) or with the seven commercial α-phosphotyrosine antibodies (Table ).

    Techniques: Infection, Expressing, Negative Control

    H tr A protein expression, oligomer formation and secretion in a large set of strains. A. Casein zymography of the indicated H . pylori strains reveals proteolytically active H tr A protein species of 55 kDa , 165 kDa and 220 kDa as labelled with arrows. Purified recombinant H tr A of 26695 is loaded in lane 9 and produced the same pattern of active HtrA forms. B. H . pylori isolates were grown in liquid BHI medium for 12 h and then fractionated in bacterial cell pellets (top) and supernatants (bottom). Immunoblotting using α‐ H tr A antibodies show that all H . pylori strains exhibit strong H tr A signals in the supernatant fraction but varied substantially in their band intensities. As control, the bacterial pellets and supernatants were probed with α‐ CagA and α‐ UreB antibodies, excluding artificially lysed bacteria.

    Journal: Molecular Microbiology

    Article Title: Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA

    doi: 10.1111/mmi.13276

    Figure Lengend Snippet: H tr A protein expression, oligomer formation and secretion in a large set of strains. A. Casein zymography of the indicated H . pylori strains reveals proteolytically active H tr A protein species of 55 kDa , 165 kDa and 220 kDa as labelled with arrows. Purified recombinant H tr A of 26695 is loaded in lane 9 and produced the same pattern of active HtrA forms. B. H . pylori isolates were grown in liquid BHI medium for 12 h and then fractionated in bacterial cell pellets (top) and supernatants (bottom). Immunoblotting using α‐ H tr A antibodies show that all H . pylori strains exhibit strong H tr A signals in the supernatant fraction but varied substantially in their band intensities. As control, the bacterial pellets and supernatants were probed with α‐ CagA and α‐ UreB antibodies, excluding artificially lysed bacteria.

    Article Snippet: CagA proteins were detected by incubation of the membranes with a rabbit polyclonal α‐CagA antibody (Austral Biologicals, USA).

    Techniques: Expressing, Zymography, Purification, Recombinant, Produced