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Santa Cruz Biotechnology antiphosphotyrosine monoclonal antibodies
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 <t>antiphosphotyrosine</t> 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.
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1) 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

2) 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

Model showing successive CagA phosphorylation, production of specific CagA PY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation. H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagA PY then inactivates c-Src in a negative feedback loop whereby direct binding of CagA PY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.
Figure Legend Snippet: Model showing successive CagA phosphorylation, production of specific CagA PY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation. H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagA PY then inactivates c-Src in a negative feedback loop whereby direct binding of CagA PY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.

Techniques Used: Infection, Binding Assay

3) Product Images from "Polymorphism in the CagA EPIYA Motif Impacts Development of Gastric Cancer ▿Polymorphism in the CagA EPIYA Motif Impacts Development of Gastric Cancer ▿ §"

Article Title: Polymorphism in the CagA EPIYA Motif Impacts Development of Gastric Cancer ▿Polymorphism in the CagA EPIYA Motif Impacts Development of Gastric Cancer ▿ §

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.02330-08

Expression, delivery, and phosphorylation of CagA. (A) Western blot analysis of bacterial lysates from the six indicated Korean strains was conducted using a monoclonal anti-CagA antibody (top) or the polyclonal anti-CagA antibody b-300 (bottom). (B)
Figure Legend Snippet: Expression, delivery, and phosphorylation of CagA. (A) Western blot analysis of bacterial lysates from the six indicated Korean strains was conducted using a monoclonal anti-CagA antibody (top) or the polyclonal anti-CagA antibody b-300 (bottom). (B)

Techniques Used: Expressing, Western Blot

4) Product Images from "Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *"

Article Title: Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.111054

STAT3 phosphorylation and nuclear localization according to the C-EPIYA status of H. pylori CagA. A , AGS cells were co-incubated with H. pylori strains for 3 h, and then their cytoplasmic and nuclear fractions were separated. The nuclear proteins were
Figure Legend Snippet: STAT3 phosphorylation and nuclear localization according to the C-EPIYA status of H. pylori CagA. A , AGS cells were co-incubated with H. pylori strains for 3 h, and then their cytoplasmic and nuclear fractions were separated. The nuclear proteins were

Techniques Used: Incubation

Relation of gp130 receptor phosphorylation to H. pylori CagA and STAT3 phosphorylation. A , the gp130 receptor was immunoprecipitated ( IP ) from the cell lysates with anti-gp130 antibody, and phosphorylated gp130 receptor was detected by immunoblotting
Figure Legend Snippet: Relation of gp130 receptor phosphorylation to H. pylori CagA and STAT3 phosphorylation. A , the gp130 receptor was immunoprecipitated ( IP ) from the cell lysates with anti-gp130 antibody, and phosphorylated gp130 receptor was detected by immunoblotting

Techniques Used: Immunoprecipitation

Effect of STAT3 activation on the migration of AGS cells co-incubated with H. pylori . The cell migration assay was performed as described under “Experimental Procedures.” AGS cells were co-incubated with H. pylori in each well of culture
Figure Legend Snippet: Effect of STAT3 activation on the migration of AGS cells co-incubated with H. pylori . The cell migration assay was performed as described under “Experimental Procedures.” AGS cells were co-incubated with H. pylori in each well of culture

Techniques Used: Activation Assay, Migration, Incubation, Cell Migration Assay

Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor
Figure Legend Snippet: Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor

Techniques Used: Incubation, Activation Assay

Relation of STAT3 activation to the induction of c- myc . A , nuclear extracts from AGS cells co-incubated with H. pylori were incubated with 32 P-labeled oligonucleotide containing the c- myc promoter E2F site and then subjected to electrophoresis and autoradiography.
Figure Legend Snippet: Relation of STAT3 activation to the induction of c- myc . A , nuclear extracts from AGS cells co-incubated with H. pylori were incubated with 32 P-labeled oligonucleotide containing the c- myc promoter E2F site and then subjected to electrophoresis and autoradiography.

Techniques Used: Activation Assay, Incubation, Labeling, Electrophoresis, Autoradiography

5) Product Images from "Functional Analysis of the cag Pathogenicity Island in Helicobacter pylori Isolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer "

Article Title: Functional Analysis of the cag Pathogenicity Island in Helicobacter pylori Isolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer

Journal: Infection and Immunity

doi: 10.1128/IAI.72.2.1043-1056.2004

Phosphorylation of the CagA protein and induction of IL-8 during infection of AGS cells with 15 representative H. pylori isolates. (A) CagA tyrosine phosphorylation was analyzed in Western blots with a phosphotyrosine-specific antibody, PY-99 (arrows). The asterisk indicates the position of an unknown 125-kDa host cell protein that is phosphorylated in the PBS control. This protein changed its phosphorylation status during infection. (B) Stripping and reprobing of the blot with an anti-CagA antibody (Ab-1) indicated the positions of the different CagA protein species on the gel (arrows). UH4, Ka223, Ka125, and Ka148/2 are cag PAI − strains and did not express CagA. CagA of strain Ca130 was expressed but not phosphorylated. Infection was for 4 h at an MOI of 100. (C) In a parallel experiment, AGS cells were infected under the same conditions for 24 h and IL-8 release into the culture supernatant was measured by ELISA. The results shown are representative of three independent experiments. The error bars indicate standard deviations.
Figure Legend Snippet: Phosphorylation of the CagA protein and induction of IL-8 during infection of AGS cells with 15 representative H. pylori isolates. (A) CagA tyrosine phosphorylation was analyzed in Western blots with a phosphotyrosine-specific antibody, PY-99 (arrows). The asterisk indicates the position of an unknown 125-kDa host cell protein that is phosphorylated in the PBS control. This protein changed its phosphorylation status during infection. (B) Stripping and reprobing of the blot with an anti-CagA antibody (Ab-1) indicated the positions of the different CagA protein species on the gel (arrows). UH4, Ka223, Ka125, and Ka148/2 are cag PAI − strains and did not express CagA. CagA of strain Ca130 was expressed but not phosphorylated. Infection was for 4 h at an MOI of 100. (C) In a parallel experiment, AGS cells were infected under the same conditions for 24 h and IL-8 release into the culture supernatant was measured by ELISA. The results shown are representative of three independent experiments. The error bars indicate standard deviations.

Techniques Used: Infection, Western Blot, Stripping Membranes, Enzyme-linked Immunosorbent Assay

Detection of unique and multiple CagA protein species. (A) Anti-phosphotyrosine patterns of AGS infected with different H. pylori ) were also observed but ran out of the gels shown. Full-length CagA is indicated by the arrowhead. The red dots indicate phosphorylated proteins corresponding in size to CagA protein species, the green dots indicate phosphorylated proteins not corresponding in size to CagA protein species, and the blue dots label CagA protein species with no detectable phosphorylated form during infection. H. pylori strain P1 served as a control because the fragmentation of 135-kDa wild-type CagA into an amino-terminal p100 CagA fragment and a carboxy-terminal p35 CagA ). +, present; −, absent. (B) H. pylori strains P284 and OM1011 are unique and showed a double band of full-length CagA. This double band had nearly identical intensities in the anti-CagA blot (black arrows) but showed a different phosphorylation pattern during infection (red arrows).
Figure Legend Snippet: Detection of unique and multiple CagA protein species. (A) Anti-phosphotyrosine patterns of AGS infected with different H. pylori ) were also observed but ran out of the gels shown. Full-length CagA is indicated by the arrowhead. The red dots indicate phosphorylated proteins corresponding in size to CagA protein species, the green dots indicate phosphorylated proteins not corresponding in size to CagA protein species, and the blue dots label CagA protein species with no detectable phosphorylated form during infection. H. pylori strain P1 served as a control because the fragmentation of 135-kDa wild-type CagA into an amino-terminal p100 CagA fragment and a carboxy-terminal p35 CagA ). +, present; −, absent. (B) H. pylori strains P284 and OM1011 are unique and showed a double band of full-length CagA. This double band had nearly identical intensities in the anti-CagA blot (black arrows) but showed a different phosphorylation pattern during infection (red arrows).

Techniques Used: Infection

Induction of c-Src inactivation and cortactin dephosphorylation. (A) CagA P-Tyr -specific inactivation of c-Src. Western blotting with a phosphospecific anti-c-Sr c antibody revealed that some, but not all, H. pylori strains induce c-Src inactivation by dephosphorylation of residue Y-418 (top) in the catalytic kinase domain. Densitometric measurements of band intensities revealed the Src activity in comparison to uninfected AGS cells in the PBS control (middle). Stripping and reprobing of the blot with a monoclonal anti-Src antibody revealed that equal amounts of Src are present in all lanes (bottom). (B) Cortactin is a major phosphorylated protein in uninfected AGS cells and is specifically dephosphorylated during infection with some H. pylori ). The phosphotyrosine pattern of cell lysates at 80 kDa shows dephosphorylation of cortactin in infections with those H. pylori isolates that induce CagA phosphorylation and Src inactivation (middle). Stripping and reprobing of the blot with a monoclonal anti-cortactin antibody revealed that similar amounts of cortactin were present in all lanes (bottom).
Figure Legend Snippet: Induction of c-Src inactivation and cortactin dephosphorylation. (A) CagA P-Tyr -specific inactivation of c-Src. Western blotting with a phosphospecific anti-c-Sr c antibody revealed that some, but not all, H. pylori strains induce c-Src inactivation by dephosphorylation of residue Y-418 (top) in the catalytic kinase domain. Densitometric measurements of band intensities revealed the Src activity in comparison to uninfected AGS cells in the PBS control (middle). Stripping and reprobing of the blot with a monoclonal anti-Src antibody revealed that equal amounts of Src are present in all lanes (bottom). (B) Cortactin is a major phosphorylated protein in uninfected AGS cells and is specifically dephosphorylated during infection with some H. pylori ). The phosphotyrosine pattern of cell lysates at 80 kDa shows dephosphorylation of cortactin in infections with those H. pylori isolates that induce CagA phosphorylation and Src inactivation (middle). Stripping and reprobing of the blot with a monoclonal anti-cortactin antibody revealed that similar amounts of cortactin were present in all lanes (bottom).

Techniques Used: De-Phosphorylation Assay, Western Blot, Activity Assay, Stripping Membranes, Infection

6) Product Images from "Inhibitory Effects of Anthocyanins on Secretion of Helicobacter pylori CagA and VacA Toxins"

Article Title: Inhibitory Effects of Anthocyanins on Secretion of Helicobacter pylori CagA and VacA Toxins

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.5094

Effect of anthocyanins on secretion of H. pylori CagA and VacA. H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.
Figure Legend Snippet: Effect of anthocyanins on secretion of H. pylori CagA and VacA. H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.

Techniques Used: Cell Culture, Western Blot

7) Product Images from "Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells"

Article Title: Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000603

Transfection of gastric epithelial cells with GFP-Bcl2 inhibits VacA-induced apoptosis while not blocking pinocytosis or intracellular trafficking of the toxin. (A) Apoptosis (shown as fold increase over the respective non-transfected control cells) induced by either VacA or etoposide in AGS or MKN 28 cells transfected with GFP-Bcl2, GFP-Bcl-xL, GFP-Mcl1, or the GFP vector. Cells were treated with VacA or etoposide as in Figure 3A . Non-treated cells served as controls. Mean±SEM of 3 independent experiments. *: P
Figure Legend Snippet: Transfection of gastric epithelial cells with GFP-Bcl2 inhibits VacA-induced apoptosis while not blocking pinocytosis or intracellular trafficking of the toxin. (A) Apoptosis (shown as fold increase over the respective non-transfected control cells) induced by either VacA or etoposide in AGS or MKN 28 cells transfected with GFP-Bcl2, GFP-Bcl-xL, GFP-Mcl1, or the GFP vector. Cells were treated with VacA or etoposide as in Figure 3A . Non-treated cells served as controls. Mean±SEM of 3 independent experiments. *: P

Techniques Used: Transfection, Blocking Assay, Plasmid Preparation

8) Product Images from "Lipoprotein Processing and Sorting in Helicobacter pylori"

Article Title: Lipoprotein Processing and Sorting in Helicobacter pylori

Journal: mBio

doi: 10.1128/mBio.00911-20

Requirement of a lipobox cysteine residue for CagT stability. (A) Amino-terminal amino acid sequences of CagT and mutant forms of CagT analyzed in this study. The CagT lipobox is highlighted in red, and the disrupted lipobox (CagT-C21S) is highlighted in blue. (B) Expression of CagT was evaluated by immunoblotting extracts of strains 26695 (WT), BV199 (Δ cagT ), BV321 (in which a WT copy of cagT was introduced into the ureA locus-restored cagT strain), BV218 ( cagT-C21S ), and BV260 ( cagT1 ) using anti-CagT. Vertical line indicates cropping of an additional unreported lane from the image. (C) H. pylori strains were cocultured with AGS cells, and the ability of each strain to induce IL-8 production was determined by ELISA. Asterisks denote results that were significantly different from the BV199 control results (ANOVA followed by Dunnett’s post hoc test, P
Figure Legend Snippet: Requirement of a lipobox cysteine residue for CagT stability. (A) Amino-terminal amino acid sequences of CagT and mutant forms of CagT analyzed in this study. The CagT lipobox is highlighted in red, and the disrupted lipobox (CagT-C21S) is highlighted in blue. (B) Expression of CagT was evaluated by immunoblotting extracts of strains 26695 (WT), BV199 (Δ cagT ), BV321 (in which a WT copy of cagT was introduced into the ureA locus-restored cagT strain), BV218 ( cagT-C21S ), and BV260 ( cagT1 ) using anti-CagT. Vertical line indicates cropping of an additional unreported lane from the image. (C) H. pylori strains were cocultured with AGS cells, and the ability of each strain to induce IL-8 production was determined by ELISA. Asterisks denote results that were significantly different from the BV199 control results (ANOVA followed by Dunnett’s post hoc test, P

Techniques Used: Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay

Analyses of CagT-DDK. (A) Amino-terminal amino acid sequence of CagT-DDK and the CagT-DDK peptides following enterokinase treatment. The lipobox is highlighted in red, the DDK epitope is underlined, and the asterisk indicates the site of triacyl lipid modification in WT H. pylori or the site of diacyl lipid modification in lnt mutant H. pylori . Signal peptide cleavage occurs between the “A” and “C” within the lipobox. Enterokinase cleavage occurs after lysine at its cleavage site DDDDK. (B) Expression of CagT was assessed by immunoblotting extracts of strains 26695 (WT), BV199 (Δ cagT ), BV321 (restored cagT ), and BV357 ( cagT-DDK 27 ) using anti-CagT and anti-HspB (as loading control). (C) H. pylori strains were cocultured with AGS cells, and the ability of each strain to induce IL-8 production was determined by ELISA. Asterisks denote results that were significantly different from the BV199 control results (analysis of variance [ANOVA] followed by Dunnett’s post hoc test, P
Figure Legend Snippet: Analyses of CagT-DDK. (A) Amino-terminal amino acid sequence of CagT-DDK and the CagT-DDK peptides following enterokinase treatment. The lipobox is highlighted in red, the DDK epitope is underlined, and the asterisk indicates the site of triacyl lipid modification in WT H. pylori or the site of diacyl lipid modification in lnt mutant H. pylori . Signal peptide cleavage occurs between the “A” and “C” within the lipobox. Enterokinase cleavage occurs after lysine at its cleavage site DDDDK. (B) Expression of CagT was assessed by immunoblotting extracts of strains 26695 (WT), BV199 (Δ cagT ), BV321 (restored cagT ), and BV357 ( cagT-DDK 27 ) using anti-CagT and anti-HspB (as loading control). (C) H. pylori strains were cocultured with AGS cells, and the ability of each strain to induce IL-8 production was determined by ELISA. Asterisks denote results that were significantly different from the BV199 control results (analysis of variance [ANOVA] followed by Dunnett’s post hoc test, P

Techniques Used: Sequencing, Modification, Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay

Activity of diacylated CagT. (A) Whole-cell lysates from H. pylori strains 26695, 26695 ΔPAI, and VM211 (Δ lnt ) were analyzed by immunoblotting with anti-CagT antisera. A representative blot is shown. (B and C) AGS cells were cultured alone or in the presence of H. pylori strains at an MOI of 100:1 for 7 h. Cell culture supernatants were analyzed for IL-8 by ELISA (B), and cell lysates were analyzed by immunoblotting (C) using an antibody recognizing phospho-Tyr (PY99) to detect phosphorylated CagA (indicated by an arrowhead) or antiserum directed against CagA (to detect total CagA). Multiple additional bands (unrelated to CagA) were detected by the anti-phospho-Tyr antibody in all samples, including AGS cells alone. Results in panel B represent means and standard deviations of three biological replicates, each analyzed in triplicate; results in panel C are representative of three biological replicates. Asterisks denote results that were significantly different from the 26695 ΔPAI control results (ANOVA followed by Dunnett’s post hoc test, P
Figure Legend Snippet: Activity of diacylated CagT. (A) Whole-cell lysates from H. pylori strains 26695, 26695 ΔPAI, and VM211 (Δ lnt ) were analyzed by immunoblotting with anti-CagT antisera. A representative blot is shown. (B and C) AGS cells were cultured alone or in the presence of H. pylori strains at an MOI of 100:1 for 7 h. Cell culture supernatants were analyzed for IL-8 by ELISA (B), and cell lysates were analyzed by immunoblotting (C) using an antibody recognizing phospho-Tyr (PY99) to detect phosphorylated CagA (indicated by an arrowhead) or antiserum directed against CagA (to detect total CagA). Multiple additional bands (unrelated to CagA) were detected by the anti-phospho-Tyr antibody in all samples, including AGS cells alone. Results in panel B represent means and standard deviations of three biological replicates, each analyzed in triplicate; results in panel C are representative of three biological replicates. Asterisks denote results that were significantly different from the 26695 ΔPAI control results (ANOVA followed by Dunnett’s post hoc test, P

Techniques Used: Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

9) Product Images from "Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer"

Article Title: Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146521

Introduction of CagA into gastric cancer cells. (A and B) Western blot analysis of CagA and phosphorylated CagA in H . pylori -infected SGC-7901(A) and AGS (B) cells. The cells infected with the indicated ratio of cells to H . pylori for the indicated time were collected and lysed, and the proteins were separated by SDS-PAGE. Cells infected with H . pylori boiled for 15 min at a MOI of 1:1000 were used as a control. (C and D) Detection of CagA mRNA and protein in cagA -overexpressing SGC-7901 cells by RT-PCR (C) and western blot (D). GAPDH served as the loading control. The data are representative of three independent experiments. Hp , H . pylori ; P-CagA, phosphorylated CagA; GAPDH, Glyceraldehyde-3-phosphate- dehydrogenase.
Figure Legend Snippet: Introduction of CagA into gastric cancer cells. (A and B) Western blot analysis of CagA and phosphorylated CagA in H . pylori -infected SGC-7901(A) and AGS (B) cells. The cells infected with the indicated ratio of cells to H . pylori for the indicated time were collected and lysed, and the proteins were separated by SDS-PAGE. Cells infected with H . pylori boiled for 15 min at a MOI of 1:1000 were used as a control. (C and D) Detection of CagA mRNA and protein in cagA -overexpressing SGC-7901 cells by RT-PCR (C) and western blot (D). GAPDH served as the loading control. The data are representative of three independent experiments. Hp , H . pylori ; P-CagA, phosphorylated CagA; GAPDH, Glyceraldehyde-3-phosphate- dehydrogenase.

Techniques Used: Western Blot, Infection, SDS Page, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "Inhibitory Effects of Anthocyanins on Secretion of Helicobacter pylori CagA and VacA Toxins"

Article Title: Inhibitory Effects of Anthocyanins on Secretion of Helicobacter pylori CagA and VacA Toxins

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.5094

Effect of anthocyanins on secretion of H. pylori CagA and VacA. H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.
Figure Legend Snippet: Effect of anthocyanins on secretion of H. pylori CagA and VacA. H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.

Techniques Used: Cell Culture, Western Blot

11) Product Images from "Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *"

Article Title: Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.111054

Effect of inhibition of gp130 or JAK2 on CagA-induced STAT activation. A , the AGS cells pretreated overnight in serum-free medium with anti-human IL6R and gp130 neutralizing monoclonal antibodies were co-incubated with H. pylori cells. Whole cell lysates
Figure Legend Snippet: Effect of inhibition of gp130 or JAK2 on CagA-induced STAT activation. A , the AGS cells pretreated overnight in serum-free medium with anti-human IL6R and gp130 neutralizing monoclonal antibodies were co-incubated with H. pylori cells. Whole cell lysates

Techniques Used: Inhibition, Activation Assay, Incubation

12) Product Images from "Lipoprotein Processing and Sorting in Helicobacter pylori"

Article Title: Lipoprotein Processing and Sorting in Helicobacter pylori

Journal: mBio

doi: 10.1128/mBio.00911-20

Activity of diacylated CagT. (A) Whole-cell lysates from H. pylori strains 26695, 26695 ΔPAI, and VM211 (Δ lnt ) were analyzed by immunoblotting with anti-CagT antisera. A representative blot is shown. (B and C) AGS cells were cultured alone or in the presence of H. pylori strains at an MOI of 100:1 for 7 h. Cell culture supernatants were analyzed for IL-8 by ELISA (B), and cell lysates were analyzed by immunoblotting (C) using an antibody recognizing phospho-Tyr (PY99) to detect phosphorylated CagA (indicated by an arrowhead) or antiserum directed against CagA (to detect total CagA). Multiple additional bands (unrelated to CagA) were detected by the anti-phospho-Tyr antibody in all samples, including AGS cells alone. Results in panel B represent means and standard deviations of three biological replicates, each analyzed in triplicate; results in panel C are representative of three biological replicates. Asterisks denote results that were significantly different from the 26695 ΔPAI control results (ANOVA followed by Dunnett’s post hoc test, P
Figure Legend Snippet: Activity of diacylated CagT. (A) Whole-cell lysates from H. pylori strains 26695, 26695 ΔPAI, and VM211 (Δ lnt ) were analyzed by immunoblotting with anti-CagT antisera. A representative blot is shown. (B and C) AGS cells were cultured alone or in the presence of H. pylori strains at an MOI of 100:1 for 7 h. Cell culture supernatants were analyzed for IL-8 by ELISA (B), and cell lysates were analyzed by immunoblotting (C) using an antibody recognizing phospho-Tyr (PY99) to detect phosphorylated CagA (indicated by an arrowhead) or antiserum directed against CagA (to detect total CagA). Multiple additional bands (unrelated to CagA) were detected by the anti-phospho-Tyr antibody in all samples, including AGS cells alone. Results in panel B represent means and standard deviations of three biological replicates, each analyzed in triplicate; results in panel C are representative of three biological replicates. Asterisks denote results that were significantly different from the 26695 ΔPAI control results (ANOVA followed by Dunnett’s post hoc test, P

Techniques Used: Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

13) Product Images from "Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *"

Article Title: Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.111054

STAT3 phosphorylation and nuclear localization according to the C-EPIYA status of H. pylori CagA. A , AGS cells were co-incubated with H. pylori strains for 3 h, and then their cytoplasmic and nuclear fractions were separated. The nuclear proteins were
Figure Legend Snippet: STAT3 phosphorylation and nuclear localization according to the C-EPIYA status of H. pylori CagA. A , AGS cells were co-incubated with H. pylori strains for 3 h, and then their cytoplasmic and nuclear fractions were separated. The nuclear proteins were

Techniques Used: Incubation

Relation of gp130 receptor phosphorylation to H. pylori CagA and STAT3 phosphorylation. A , the gp130 receptor was immunoprecipitated ( IP ) from the cell lysates with anti-gp130 antibody, and phosphorylated gp130 receptor was detected by immunoblotting
Figure Legend Snippet: Relation of gp130 receptor phosphorylation to H. pylori CagA and STAT3 phosphorylation. A , the gp130 receptor was immunoprecipitated ( IP ) from the cell lysates with anti-gp130 antibody, and phosphorylated gp130 receptor was detected by immunoblotting

Techniques Used: Immunoprecipitation

Effect of STAT3 activation on the migration of AGS cells co-incubated with H. pylori . The cell migration assay was performed as described under “Experimental Procedures.” AGS cells were co-incubated with H. pylori in each well of culture
Figure Legend Snippet: Effect of STAT3 activation on the migration of AGS cells co-incubated with H. pylori . The cell migration assay was performed as described under “Experimental Procedures.” AGS cells were co-incubated with H. pylori in each well of culture

Techniques Used: Activation Assay, Migration, Incubation, Cell Migration Assay

Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor
Figure Legend Snippet: Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor

Techniques Used: Incubation, Activation Assay

Relation of STAT3 activation to the induction of c- myc . A , nuclear extracts from AGS cells co-incubated with H. pylori were incubated with 32 P-labeled oligonucleotide containing the c- myc promoter E2F site and then subjected to electrophoresis and autoradiography.
Figure Legend Snippet: Relation of STAT3 activation to the induction of c- myc . A , nuclear extracts from AGS cells co-incubated with H. pylori were incubated with 32 P-labeled oligonucleotide containing the c- myc promoter E2F site and then subjected to electrophoresis and autoradiography.

Techniques Used: Activation Assay, Incubation, Labeling, Electrophoresis, Autoradiography

14) Product Images from "Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *"

Article Title: Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.111054

Relationship between the C-EPIYA status of H. pylori CagA and its interaction with SHP2 and gp130. A , lysates of AGS cells co-incubated with H. pylori were immunoprecipitated ( IP ) with anti-SHP2 or anti-CagA antibody and then immunoblotted ( IB ) with the
Figure Legend Snippet: Relationship between the C-EPIYA status of H. pylori CagA and its interaction with SHP2 and gp130. A , lysates of AGS cells co-incubated with H. pylori were immunoprecipitated ( IP ) with anti-SHP2 or anti-CagA antibody and then immunoblotted ( IB ) with the

Techniques Used: Incubation, Immunoprecipitation

Relation of gp130 receptor phosphorylation to H. pylori CagA and STAT3 phosphorylation. A , the gp130 receptor was immunoprecipitated ( IP ) from the cell lysates with anti-gp130 antibody, and phosphorylated gp130 receptor was detected by immunoblotting
Figure Legend Snippet: Relation of gp130 receptor phosphorylation to H. pylori CagA and STAT3 phosphorylation. A , the gp130 receptor was immunoprecipitated ( IP ) from the cell lysates with anti-gp130 antibody, and phosphorylated gp130 receptor was detected by immunoblotting

Techniques Used: Immunoprecipitation

Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor
Figure Legend Snippet: Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor

Techniques Used: Incubation, Activation Assay

Effect of inhibition of gp130 or JAK2 on CagA-induced STAT activation. A , the AGS cells pretreated overnight in serum-free medium with anti-human IL6R and gp130 neutralizing monoclonal antibodies were co-incubated with H. pylori cells. Whole cell lysates
Figure Legend Snippet: Effect of inhibition of gp130 or JAK2 on CagA-induced STAT activation. A , the AGS cells pretreated overnight in serum-free medium with anti-human IL6R and gp130 neutralizing monoclonal antibodies were co-incubated with H. pylori cells. Whole cell lysates

Techniques Used: Inhibition, Activation Assay, Incubation

15) Product Images from "Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells"

Article Title: Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000603

Transfection of gastric epithelial cells with GFP-Bcl2 inhibits VacA-induced apoptosis while not blocking pinocytosis or intracellular trafficking of the toxin. (A) Apoptosis (shown as fold increase over the respective non-transfected control cells) induced by either VacA or etoposide in AGS or MKN 28 cells transfected with GFP-Bcl2, GFP-Bcl-xL, GFP-Mcl1, or the GFP vector. Cells were treated with VacA or etoposide as in Figure 3A . Non-treated cells served as controls. Mean±SEM of 3 independent experiments. *: P
Figure Legend Snippet: Transfection of gastric epithelial cells with GFP-Bcl2 inhibits VacA-induced apoptosis while not blocking pinocytosis or intracellular trafficking of the toxin. (A) Apoptosis (shown as fold increase over the respective non-transfected control cells) induced by either VacA or etoposide in AGS or MKN 28 cells transfected with GFP-Bcl2, GFP-Bcl-xL, GFP-Mcl1, or the GFP vector. Cells were treated with VacA or etoposide as in Figure 3A . Non-treated cells served as controls. Mean±SEM of 3 independent experiments. *: P

Techniques Used: Transfection, Blocking Assay, Plasmid Preparation

16) 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

Model showing successive CagA phosphorylation, production of specific CagA PY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation. H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagA PY then inactivates c-Src in a negative feedback loop whereby direct binding of CagA PY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.
Figure Legend Snippet: Model showing successive CagA phosphorylation, production of specific CagA PY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation. H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagA PY then inactivates c-Src in a negative feedback loop whereby direct binding of CagA PY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.

Techniques Used: Infection, Binding Assay

17) Product Images from "Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *"

Article Title: Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.111054

Effect of inhibition of gp130 or JAK2 on CagA-induced STAT activation. A , the AGS cells pretreated overnight in serum-free medium with anti-human IL6R and gp130 neutralizing monoclonal antibodies were co-incubated with H. pylori cells. Whole cell lysates
Figure Legend Snippet: Effect of inhibition of gp130 or JAK2 on CagA-induced STAT activation. A , the AGS cells pretreated overnight in serum-free medium with anti-human IL6R and gp130 neutralizing monoclonal antibodies were co-incubated with H. pylori cells. Whole cell lysates

Techniques Used: Inhibition, Activation Assay, Incubation

18) Product Images from "Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface"

Article Title: Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002050

VacA contributes to Hp colonization of the apical cell surface. (A) VacA aids Hp colonization of the apical cell surface. Using the Transwell system, cells were infected with WT or Δ vacA , and co-culture media added only to the basal chamber (+). DMEM was added to the apical chamber (−). Samples were taken daily from the apical chamber and plated for CFU counts. (B) Exogenous addition of iron apically rescues Δ vacA growth on the polarized epithelium. Polarized cells were infected as in (A). Solid lines indicate conditions with DMEM apically. Dashed lines indicate conditions with 100 µM ferric chloride (Fe 3+ ) added to the apical DMEM. Samples were taken and plated as in (A). (C) 3D confocal images of WT or Δ vacA colonizing the cell surface of polarized MDCK cells in the Transwell system. Cells were infected for 5 minutes and then unattached bacteria washed away and media replaced. At 1, 3 and 5 days post-infection, samples were fixed and processed for immunofluorescence. Bacteria are visualized with anti- Hp antibodies (green) and cell junctions are stained red (anti-ZO-1). Scale bar 10 µm. (D) Quantification of WT, Δ vacA and VacA* microcolony sizes over time (1, 3 and 5 days), determined by fluorescence volume measured from multiple 3D confocal images. VacA* is the complemented Δ vacA mutant. Each point on the graph represents a microcolony. p-value was obtained with a Mann-Whitney statistical test. (E) CagA and VacA work in concert to enable Hp colonization of the polarized epithelium. Polarized cells were infected as in (A) with the strains indicated. Samples were taken and plated as in (A). (F) CagA and VacA work together to aid Hp acquisition of iron from host cells. Polarized cells were infected as in (A). DMEM (solid lines) or DMEM + 100 µM ferric chloride (Fe 3+ , dashed lines) was added apically. Samples were taken and plated as in (A).
Figure Legend Snippet: VacA contributes to Hp colonization of the apical cell surface. (A) VacA aids Hp colonization of the apical cell surface. Using the Transwell system, cells were infected with WT or Δ vacA , and co-culture media added only to the basal chamber (+). DMEM was added to the apical chamber (−). Samples were taken daily from the apical chamber and plated for CFU counts. (B) Exogenous addition of iron apically rescues Δ vacA growth on the polarized epithelium. Polarized cells were infected as in (A). Solid lines indicate conditions with DMEM apically. Dashed lines indicate conditions with 100 µM ferric chloride (Fe 3+ ) added to the apical DMEM. Samples were taken and plated as in (A). (C) 3D confocal images of WT or Δ vacA colonizing the cell surface of polarized MDCK cells in the Transwell system. Cells were infected for 5 minutes and then unattached bacteria washed away and media replaced. At 1, 3 and 5 days post-infection, samples were fixed and processed for immunofluorescence. Bacteria are visualized with anti- Hp antibodies (green) and cell junctions are stained red (anti-ZO-1). Scale bar 10 µm. (D) Quantification of WT, Δ vacA and VacA* microcolony sizes over time (1, 3 and 5 days), determined by fluorescence volume measured from multiple 3D confocal images. VacA* is the complemented Δ vacA mutant. Each point on the graph represents a microcolony. p-value was obtained with a Mann-Whitney statistical test. (E) CagA and VacA work in concert to enable Hp colonization of the polarized epithelium. Polarized cells were infected as in (A) with the strains indicated. Samples were taken and plated as in (A). (F) CagA and VacA work together to aid Hp acquisition of iron from host cells. Polarized cells were infected as in (A). DMEM (solid lines) or DMEM + 100 µM ferric chloride (Fe 3+ , dashed lines) was added apically. Samples were taken and plated as in (A).

Techniques Used: Infection, Co-Culture Assay, Immunofluorescence, Staining, Fluorescence, Mutagenesis, MANN-WHITNEY

Hp colonization of the apical cell surface increases internalized transferrin. (A) 3D confocal images of fluorescent transferrin signal in polarized MDCK cells stably expressing human transferrin receptor, uninfected or infected with WT or Δ cagA . Fluorescent transferrin was added to the basal chamber and incubated on ice for 30 minutes, unbound transferrin washed away, then immediately fixed (top panels, cross-sectional view, 0 minutes post-uptake), or further incubated for 30 minutes at 37°C to allow uptake of bound transferrin (middle and bottom panels, top and cross-sectional views, 30 minutes post-uptake). Fluorescent transferrin is shown in red, and bacteria are visualized with anti- Hp antibodies (blue). Scale bars 10 µm. (B) Hp colonization does not significantly affect host cell transferrin receptor expression. Polarized cells in the Transwell system were infected for 2 days with WT or Δ cagA . Whole-cell lysates from these infections were separated by SDS-PAGE, transferred to a nitrocellulose membrane, then immunoblotted with antibodies against transferrin receptor (top panel) and against GAPDH as a loading control (bottom panel). (C) Quantification of transferrin fluorescence 30 minutes post-uptake in polarized epithelial monolayers. Monolayers were infected for 2 days with the indicated Hp strains, fixed after 30 minutes of transferrin uptake, and total transferrin fluorescence measured from multiple 3D confocal images. EPISA is a mutant expressing a mutated CagA that cannot be phosphorylated. CagA* is the complemented Δ cagA mutant. p-values were obtained with a Mann-Whitney statistical test. N.S. indicates no statistical significance.
Figure Legend Snippet: Hp colonization of the apical cell surface increases internalized transferrin. (A) 3D confocal images of fluorescent transferrin signal in polarized MDCK cells stably expressing human transferrin receptor, uninfected or infected with WT or Δ cagA . Fluorescent transferrin was added to the basal chamber and incubated on ice for 30 minutes, unbound transferrin washed away, then immediately fixed (top panels, cross-sectional view, 0 minutes post-uptake), or further incubated for 30 minutes at 37°C to allow uptake of bound transferrin (middle and bottom panels, top and cross-sectional views, 30 minutes post-uptake). Fluorescent transferrin is shown in red, and bacteria are visualized with anti- Hp antibodies (blue). Scale bars 10 µm. (B) Hp colonization does not significantly affect host cell transferrin receptor expression. Polarized cells in the Transwell system were infected for 2 days with WT or Δ cagA . Whole-cell lysates from these infections were separated by SDS-PAGE, transferred to a nitrocellulose membrane, then immunoblotted with antibodies against transferrin receptor (top panel) and against GAPDH as a loading control (bottom panel). (C) Quantification of transferrin fluorescence 30 minutes post-uptake in polarized epithelial monolayers. Monolayers were infected for 2 days with the indicated Hp strains, fixed after 30 minutes of transferrin uptake, and total transferrin fluorescence measured from multiple 3D confocal images. EPISA is a mutant expressing a mutated CagA that cannot be phosphorylated. CagA* is the complemented Δ cagA mutant. p-values were obtained with a Mann-Whitney statistical test. N.S. indicates no statistical significance.

Techniques Used: Stable Transfection, Expressing, Infection, Incubation, SDS Page, Fluorescence, Mutagenesis, MANN-WHITNEY

Transferrin receptor is mislocalized apically to sites of bacterial microcolonies. (A) Polarized MDCK cells in the Transwell system were infected with WT, Δ cagA , or Δ vacA for 2 days. Apical staining with anti-transferrin receptor antibodies was carried out on non-permeabilized samples. Bacteria are visualized with anti- Hp antibodies (blue), transferrin receptor (tfR) is stained red, and phalloidin staining of f-actin is shown in yellow. 3D confocal images are shown, and cross-sectional view is also presented for WT (second row). Scale bars 5 µm. (B) Quantitative data of the transferrin receptor (tfR) fluorescence intensity associated with the bacterial microcolonies, determined by the fluorescence voxel volume of each microcolony stained with anti- Hp antibodies, and the fluorescence sum of the transferrin receptor signal associated with the microcolonies, measured from multiple 3D confocal images. VacA* is the complemented Δ vacA mutant. Each point on the graph represents a microcolony. p-values were obtained with a Mann-Whitney statistical test. N.S. indicates no statistical significance.
Figure Legend Snippet: Transferrin receptor is mislocalized apically to sites of bacterial microcolonies. (A) Polarized MDCK cells in the Transwell system were infected with WT, Δ cagA , or Δ vacA for 2 days. Apical staining with anti-transferrin receptor antibodies was carried out on non-permeabilized samples. Bacteria are visualized with anti- Hp antibodies (blue), transferrin receptor (tfR) is stained red, and phalloidin staining of f-actin is shown in yellow. 3D confocal images are shown, and cross-sectional view is also presented for WT (second row). Scale bars 5 µm. (B) Quantitative data of the transferrin receptor (tfR) fluorescence intensity associated with the bacterial microcolonies, determined by the fluorescence voxel volume of each microcolony stained with anti- Hp antibodies, and the fluorescence sum of the transferrin receptor signal associated with the microcolonies, measured from multiple 3D confocal images. VacA* is the complemented Δ vacA mutant. Each point on the graph represents a microcolony. p-values were obtained with a Mann-Whitney statistical test. N.S. indicates no statistical significance.

Techniques Used: Infection, Staining, Fluorescence, Mutagenesis, MANN-WHITNEY

19) Product Images from "Functional Analysis of the cag Pathogenicity Island in Helicobacter pylori Isolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer "

Article Title: Functional Analysis of the cag Pathogenicity Island in Helicobacter pylori Isolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer

Journal: Infection and Immunity

doi: 10.1128/IAI.72.2.1043-1056.2004

In vitro phosphorylation of CagA. (A) We observed nine nonphosphorylated CagA protein species during infection of AGS cells with the following H. pylori strains: M15, M27, M33, and M64 (gastritis group); Ka61 (peptic ulcer group); and MPI47, Ca115, Ca130, and Ca204 (gastric cancer group). These CagA proteins could be phosphorylated in an in vitro phosphorylation assay. The results are shown for three strains. Incubation of the H. pylori lysate with lysate of AGS cells or recombinant human c-Src (data not shown) resulted in specific CagA phosphorylation (arrowhead). The lysates of the TIGR H. pylori strain 26695 and a phosphorylation-deficient deletion mutant (ΔP-Tyr) were used as positive and negative controls, respectively. CagA phosphorylation was not detected either in the lysate of wild-type H. pylori incubated without c-Src or in the lysate of the CagAΔP-Tyr mutant incubated with c-Src. (B) An anti-CagA blot with antibody Ab-1 was performed as a control. +, present; −, absent.
Figure Legend Snippet: In vitro phosphorylation of CagA. (A) We observed nine nonphosphorylated CagA protein species during infection of AGS cells with the following H. pylori strains: M15, M27, M33, and M64 (gastritis group); Ka61 (peptic ulcer group); and MPI47, Ca115, Ca130, and Ca204 (gastric cancer group). These CagA proteins could be phosphorylated in an in vitro phosphorylation assay. The results are shown for three strains. Incubation of the H. pylori lysate with lysate of AGS cells or recombinant human c-Src (data not shown) resulted in specific CagA phosphorylation (arrowhead). The lysates of the TIGR H. pylori strain 26695 and a phosphorylation-deficient deletion mutant (ΔP-Tyr) were used as positive and negative controls, respectively. CagA phosphorylation was not detected either in the lysate of wild-type H. pylori incubated without c-Src or in the lysate of the CagAΔP-Tyr mutant incubated with c-Src. (B) An anti-CagA blot with antibody Ab-1 was performed as a control. +, present; −, absent.

Techniques Used: In Vitro, Infection, Phosphorylation Assay, Incubation, Recombinant, Mutagenesis

20) Product Images from "Caveolin-1 Protects B6129 Mice against Helicobacter pylori Gastritis"

Article Title: Caveolin-1 Protects B6129 Mice against Helicobacter pylori Gastritis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003251

Loss of Cav1 promotes recruitment of macrophages to stomachs infected with H. pylori SS1. (A) Differential expression of mRNAs in mouse gastric tissue upon an 11-month infection with H. pylori strain SS1. CT-values from RT-qPCRs on total RNA extracted from resected stomachs were normalized to beta-2-microglobulin (b2M) and are presented as mean ± S.E. (n = 15 per group); *p
Figure Legend Snippet: Loss of Cav1 promotes recruitment of macrophages to stomachs infected with H. pylori SS1. (A) Differential expression of mRNAs in mouse gastric tissue upon an 11-month infection with H. pylori strain SS1. CT-values from RT-qPCRs on total RNA extracted from resected stomachs were normalized to beta-2-microglobulin (b2M) and are presented as mean ± S.E. (n = 15 per group); *p

Techniques Used: Infection, Expressing

21) Product Images from "Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *"

Article Title: Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.111054

Relationship between the C-EPIYA status of H. pylori CagA and its interaction with SHP2 and gp130. A , lysates of AGS cells co-incubated with H. pylori were immunoprecipitated ( IP ) with anti-SHP2 or anti-CagA antibody and then immunoblotted ( IB ) with the
Figure Legend Snippet: Relationship between the C-EPIYA status of H. pylori CagA and its interaction with SHP2 and gp130. A , lysates of AGS cells co-incubated with H. pylori were immunoprecipitated ( IP ) with anti-SHP2 or anti-CagA antibody and then immunoblotted ( IB ) with the

Techniques Used: Incubation, Immunoprecipitation

SHP2/ERK activation according to H. pylori CagA status. A , lysates from AGS cells co-incubated with H. pylori strains were immunoblotted with anti-p-SHP2, and then the membrane was stripped and analyzed with anti-SHP2 antibody to standardize gel loading.
Figure Legend Snippet: SHP2/ERK activation according to H. pylori CagA status. A , lysates from AGS cells co-incubated with H. pylori strains were immunoblotted with anti-p-SHP2, and then the membrane was stripped and analyzed with anti-SHP2 antibody to standardize gel loading.

Techniques Used: Activation Assay, Incubation

Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor
Figure Legend Snippet: Proposed role of CagA in signal switching between the JAK/STAT3 and SHP2/ERK pathways via the gp130 receptor in AGS cells. AGS cells incubated with CagA + EPIYA − H. pylori have preferential activation of the JAK/STAT3 pathway, via the IL6 receptor

Techniques Used: Incubation, Activation Assay

22) Product Images from "KCTD5 and Ubiquitin Proteasome Signaling Are Required for Helicobacter pylori Adherence"

Article Title: KCTD5 and Ubiquitin Proteasome Signaling Are Required for Helicobacter pylori Adherence

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00450

Ubiquitination of total proteins and proteasome activity in AGS cells co-cultured with H. pylori . (A) Representative immunoblot of total ubiquitinated protein from AGS cells infected with H. pylori (MOI = 300) for 8 h, in presence or absence of 2.5 μM Lactacystin. Tubulin is shown as loading control and CagA as infection control. (B) Quantification of proteasome activity in AGS cells infected with H. pylori wild type and its isogenic mutants cagA, vacA and, in presence of 2.5 μM Lactacystin for 8 h. Asterisk represents significant difference ( p
Figure Legend Snippet: Ubiquitination of total proteins and proteasome activity in AGS cells co-cultured with H. pylori . (A) Representative immunoblot of total ubiquitinated protein from AGS cells infected with H. pylori (MOI = 300) for 8 h, in presence or absence of 2.5 μM Lactacystin. Tubulin is shown as loading control and CagA as infection control. (B) Quantification of proteasome activity in AGS cells infected with H. pylori wild type and its isogenic mutants cagA, vacA and, in presence of 2.5 μM Lactacystin for 8 h. Asterisk represents significant difference ( p

Techniques Used: Activity Assay, Cell Culture, Infection

23) Product Images from "Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease"

Article Title: Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease

Journal: mBio

doi: 10.1128/mBio.01779-16

Relative levels of CagA protein and CagA phosphorylation. (A) The protein levels of CagA and UreA in lysates of H. pylori strains PMSS1, S F -1, S L -2, M F -3, and M L -1 were measured by Western blotting (upper panel). For each lysate, 0.5, 1, 1.5, and 2 μg of total protein were used to determine standard curves for CagA and UreA. The immunoblot images were analyzed using ImageJ software, and the values were plotted on a graph (lower panel). (B) Ratios of CagA to UreA were calculated, and each value was normalized to the value calculated for cagA -S F -1 to determine relative CagA protein levels. The bar graphs indicate average levels of CagA expression of each strain, and error bars represent standard deviations, derived from results of 2 independent experiments. (C) Lysates of AGS cells that were infected with H. pylori strains PMSS1, Δ cagA FL -2, S F -1, S L -2, M F -3, and M L -1 were immunoblotted for phosphorylated CagA (p-CagA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CagA, and UreA. GAPDH and UreA were used as controls.
Figure Legend Snippet: Relative levels of CagA protein and CagA phosphorylation. (A) The protein levels of CagA and UreA in lysates of H. pylori strains PMSS1, S F -1, S L -2, M F -3, and M L -1 were measured by Western blotting (upper panel). For each lysate, 0.5, 1, 1.5, and 2 μg of total protein were used to determine standard curves for CagA and UreA. The immunoblot images were analyzed using ImageJ software, and the values were plotted on a graph (lower panel). (B) Ratios of CagA to UreA were calculated, and each value was normalized to the value calculated for cagA -S F -1 to determine relative CagA protein levels. The bar graphs indicate average levels of CagA expression of each strain, and error bars represent standard deviations, derived from results of 2 independent experiments. (C) Lysates of AGS cells that were infected with H. pylori strains PMSS1, Δ cagA FL -2, S F -1, S L -2, M F -3, and M L -1 were immunoblotted for phosphorylated CagA (p-CagA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CagA, and UreA. GAPDH and UreA were used as controls.

Techniques Used: Western Blot, Software, Expressing, Derivative Assay, Infection

24) 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

25) Product Images from "Proteasome Particle-Rich Structures Are Widely Present in Human Epithelial Neoplasms: Correlative Light, Confocal and Electron Microscopy Study"

Article Title: Proteasome Particle-Rich Structures Are Widely Present in Human Epithelial Neoplasms: Correlative Light, Confocal and Electron Microscopy Study

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021317

Lung large cell carcinoma, hepatocarcinoma and colon adenocarcinoma. ( A – C ) Juxtanuclear PaCS in a lung large cell carcinoma ( A , 10,000x), boxed area enlarged in B (50,000x) whose boxed area is enlarged in C (75,000x) to show its particulate structure and proteasome immunogold reactivity. ( D and E ) Hepatocellular carcinoma showing both hyaline bodies (hb), reactive for p62 protein in D (30,000x) and unreactive for 19S proteasome in E (25,000x), and PaCS (asterisks) unreactive for p62 and reactive for 19S proteasome. r, ribosomes. Inset to D (450x): hyaline and Mallory bodies react for p62 protein immunoperoxidase in formalin-fixed paraffin sections. ( F – H ) Colon adenocarcinoma ( F , 10,000x) with partly intracellular and partly interstitial bacteria, a cytoplasmic vacuole (v) and a PaCS (boxed) bordering the cell plasma membrane, enlarged in G (40,000x) and H (75,000x). Note partial lysis of proteasome particles, though retaining 19S proteasome reactivity, development of irregular dense bodies and an encircling peripheral membrane, suggesting transition from a PaCS to an autophagic vacuole, also enclosing ribosome-like particles (r) and a bacterium (top right in G and H ) with capsulated wall typical of Gram-positive organisms.
Figure Legend Snippet: Lung large cell carcinoma, hepatocarcinoma and colon adenocarcinoma. ( A – C ) Juxtanuclear PaCS in a lung large cell carcinoma ( A , 10,000x), boxed area enlarged in B (50,000x) whose boxed area is enlarged in C (75,000x) to show its particulate structure and proteasome immunogold reactivity. ( D and E ) Hepatocellular carcinoma showing both hyaline bodies (hb), reactive for p62 protein in D (30,000x) and unreactive for 19S proteasome in E (25,000x), and PaCS (asterisks) unreactive for p62 and reactive for 19S proteasome. r, ribosomes. Inset to D (450x): hyaline and Mallory bodies react for p62 protein immunoperoxidase in formalin-fixed paraffin sections. ( F – H ) Colon adenocarcinoma ( F , 10,000x) with partly intracellular and partly interstitial bacteria, a cytoplasmic vacuole (v) and a PaCS (boxed) bordering the cell plasma membrane, enlarged in G (40,000x) and H (75,000x). Note partial lysis of proteasome particles, though retaining 19S proteasome reactivity, development of irregular dense bodies and an encircling peripheral membrane, suggesting transition from a PaCS to an autophagic vacuole, also enclosing ribosome-like particles (r) and a bacterium (top right in G and H ) with capsulated wall typical of Gram-positive organisms.

Techniques Used: Lysis

26) 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

Model showing successive CagA phosphorylation, production of specific CagA PY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation. H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagA PY then inactivates c-Src in a negative feedback loop whereby direct binding of CagA PY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.
Figure Legend Snippet: Model showing successive CagA phosphorylation, production of specific CagA PY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation. H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagA PY then inactivates c-Src in a negative feedback loop whereby direct binding of CagA PY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.

Techniques Used: Infection, Binding Assay

Related Articles

Transfection:

Article Title: Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC
Article Snippet: .. siRNA transfections and real-time PCR siRNA for β-arrestin-1, E2F1, c-Src and EGFR were purchased from Santa Cruz Biotechnology Inc. A non-targeting siRNA sequence was used as control. .. 100 pmol of siRNAs (Santa Cruz Biotechnology, USA) were transfected using Oligofectamine.

Preserving:

Article Title: Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor
Article Snippet: .. After extensive washing with membrane preserving buffer, MDCK cells were incubated with rabbit polyclonal anti-α-enolase antibody (Santa Cruz Biotechnology) (1:50 in 1%BSA/PBS) at 37 °C for 1 h. The cells were then rinsed with PBS three times and then incubated with Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) containing 0.1μg/ml Hoechst dye (DNA staining for nuclear localization) (Sigma; St. Louis, MO) at 37 °C for 1 h. Thereafter, the stained MDCK cells were washed with PBS and mounted with 50% glycerol/PBS for subsequent examination using a laser-scanning confocal microscope. .. 3-D planes of X-Y, X-Z and Y-Z scans were captured under ECLIPSE Ti-Clsi4 Laser Unit (Nikon; Tokyo, Japan) equipped with NIS-Elements D V.4.11 (Nikon).

Microscopy:

Article Title: Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor
Article Snippet: .. After extensive washing with membrane preserving buffer, MDCK cells were incubated with rabbit polyclonal anti-α-enolase antibody (Santa Cruz Biotechnology) (1:50 in 1%BSA/PBS) at 37 °C for 1 h. The cells were then rinsed with PBS three times and then incubated with Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) containing 0.1μg/ml Hoechst dye (DNA staining for nuclear localization) (Sigma; St. Louis, MO) at 37 °C for 1 h. Thereafter, the stained MDCK cells were washed with PBS and mounted with 50% glycerol/PBS for subsequent examination using a laser-scanning confocal microscope. .. 3-D planes of X-Y, X-Z and Y-Z scans were captured under ECLIPSE Ti-Clsi4 Laser Unit (Nikon; Tokyo, Japan) equipped with NIS-Elements D V.4.11 (Nikon).

Real-time Polymerase Chain Reaction:

Article Title: Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC
Article Snippet: .. siRNA transfections and real-time PCR siRNA for β-arrestin-1, E2F1, c-Src and EGFR were purchased from Santa Cruz Biotechnology Inc. A non-targeting siRNA sequence was used as control. .. 100 pmol of siRNAs (Santa Cruz Biotechnology, USA) were transfected using Oligofectamine.

Incubation:

Article Title: Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor
Article Snippet: .. After extensive washing with membrane preserving buffer, MDCK cells were incubated with rabbit polyclonal anti-α-enolase antibody (Santa Cruz Biotechnology) (1:50 in 1%BSA/PBS) at 37 °C for 1 h. The cells were then rinsed with PBS three times and then incubated with Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) containing 0.1μg/ml Hoechst dye (DNA staining for nuclear localization) (Sigma; St. Louis, MO) at 37 °C for 1 h. Thereafter, the stained MDCK cells were washed with PBS and mounted with 50% glycerol/PBS for subsequent examination using a laser-scanning confocal microscope. .. 3-D planes of X-Y, X-Z and Y-Z scans were captured under ECLIPSE Ti-Clsi4 Laser Unit (Nikon; Tokyo, Japan) equipped with NIS-Elements D V.4.11 (Nikon).

Article Title: Sensitivity Analysis of Intracellular Signaling Pathway Kinetics Predicts Targets for Stem Cell Fate Control
Article Snippet: .. The following primary antibodies were used with 24-h incubation: anti-phospho-Stat3 (tyr705) (9131; Cell Signaling Technology, http://www.cellsignal.com/ ), anti-Oct3 (Oct4) (611203; BD Bioscience, http://www.bdbiosciences.com/ ), anti-phospho-Jak2 (Tyr1007–1008) (3771; Cell Signaling technology), anti-LIFR (Sc659-Santa Cruz Biotechnology, http://www.scbt.com/ ), anti- GP130 (Sc656-Santa Cruz Biotechnology), and anti-SOCS3 (Sc9023-Santa Cruz Biotechnology). .. The following secondary antibodies were used with 2-h incubation: AlexaFluor 488 (A-11034), and AlexaFluor 546 (A-11030; Molecular Probes, http://www.probes.invitrogen.com ).

Article Title: Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution
Article Snippet: .. The bicinchoninic acid assays were subsequently performed to normalize protein levels, and cell extracts were applied to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membrane, blocked in 5% non-fat milk in Tris-buffered saline containing Tween-20 (0.9% NaCl and 0.05% Tween-20 in 20 mM Tris/HCl, pH 7.4) for 1 h, and incubated with following antibodies: HIF-1α (1:1000, NB100-479, Novus Biologicals, Littleton, CO, USA), HIF-2α (1:1000, NB100-122, Novus Biologicals), EPO (1:500, sc-7956, Santa Cruz, CA, USA), EPOR (1:500, sc-697, Santa Cruz, CA, USA), PPARγ (1:500, ab19481, Abcam, Cambridge, UK), p-Jak2 (1:500, sc-16566-R, Santa Cruz, CA, USA), Jak2 (1:500, sc-278, Santa Cruz, CA, USA), GAPDH (1:1,000, AB-P-R001, Goodhere Biotechnology, Hangzhou, China) for 1 h followed by addition of secondary antibody the goat anti-rabbit IgG-HRP secondary antibody (1:1,000, NBP1-75297, Novus Biologicals, Littleton, CO, USA) for 30 min. Blots were washed and detection was performed using an enhanced chemiluminescence system BeyoECL Plus (Beyotime Institute of Biotechnology, Haimen, China). ..

other:

Article Title: MicroRNA-21 may be involved in the therapeutic effects of Galla chinensis ointment on keloid
Article Snippet: PI3K was highly expressed in both the control and treatment groups.

Article Title: ?-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades
Article Snippet: LY294002, a potent inhibitor of PI3K was purchased from Santa Cruz Biotechnology, inc. (sc-201426).

Polyacrylamide Gel Electrophoresis:

Article Title: Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution
Article Snippet: .. The bicinchoninic acid assays were subsequently performed to normalize protein levels, and cell extracts were applied to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membrane, blocked in 5% non-fat milk in Tris-buffered saline containing Tween-20 (0.9% NaCl and 0.05% Tween-20 in 20 mM Tris/HCl, pH 7.4) for 1 h, and incubated with following antibodies: HIF-1α (1:1000, NB100-479, Novus Biologicals, Littleton, CO, USA), HIF-2α (1:1000, NB100-122, Novus Biologicals), EPO (1:500, sc-7956, Santa Cruz, CA, USA), EPOR (1:500, sc-697, Santa Cruz, CA, USA), PPARγ (1:500, ab19481, Abcam, Cambridge, UK), p-Jak2 (1:500, sc-16566-R, Santa Cruz, CA, USA), Jak2 (1:500, sc-278, Santa Cruz, CA, USA), GAPDH (1:1,000, AB-P-R001, Goodhere Biotechnology, Hangzhou, China) for 1 h followed by addition of secondary antibody the goat anti-rabbit IgG-HRP secondary antibody (1:1,000, NBP1-75297, Novus Biologicals, Littleton, CO, USA) for 30 min. Blots were washed and detection was performed using an enhanced chemiluminescence system BeyoECL Plus (Beyotime Institute of Biotechnology, Haimen, China). ..

Sequencing:

Article Title: Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC
Article Snippet: .. siRNA transfections and real-time PCR siRNA for β-arrestin-1, E2F1, c-Src and EGFR were purchased from Santa Cruz Biotechnology Inc. A non-targeting siRNA sequence was used as control. .. 100 pmol of siRNAs (Santa Cruz Biotechnology, USA) were transfected using Oligofectamine.

Staining:

Article Title: Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor
Article Snippet: .. After extensive washing with membrane preserving buffer, MDCK cells were incubated with rabbit polyclonal anti-α-enolase antibody (Santa Cruz Biotechnology) (1:50 in 1%BSA/PBS) at 37 °C for 1 h. The cells were then rinsed with PBS three times and then incubated with Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) containing 0.1μg/ml Hoechst dye (DNA staining for nuclear localization) (Sigma; St. Louis, MO) at 37 °C for 1 h. Thereafter, the stained MDCK cells were washed with PBS and mounted with 50% glycerol/PBS for subsequent examination using a laser-scanning confocal microscope. .. 3-D planes of X-Y, X-Z and Y-Z scans were captured under ECLIPSE Ti-Clsi4 Laser Unit (Nikon; Tokyo, Japan) equipped with NIS-Elements D V.4.11 (Nikon).

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  • 85
    Santa Cruz Biotechnology mouse anti caga monoclonal antibody
    Effect of anthocyanins on secretion of H. pylori <t>CagA</t> and <t>VacA.</t> H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.
    Mouse Anti Caga Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti caga monoclonal antibody/product/Santa Cruz Biotechnology
    Average 85 stars, based on 4 article reviews
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    mouse anti caga monoclonal antibody - by Bioz Stars, 2020-09
    85/100 stars
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    88
    Santa Cruz Biotechnology monoclonal antibody against caga
    Effect of anthocyanins on secretion of H. pylori <t>CagA</t> and <t>VacA.</t> H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.
    Monoclonal Antibody Against Caga, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal antibody against caga/product/Santa Cruz Biotechnology
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    Effect of anthocyanins on secretion of H. pylori CagA and VacA. H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.

    Journal: International Journal of Medical Sciences

    Article Title: Inhibitory Effects of Anthocyanins on Secretion of Helicobacter pylori CagA and VacA Toxins

    doi: 10.7150/ijms.5094

    Figure Lengend Snippet: Effect of anthocyanins on secretion of H. pylori CagA and VacA. H. pylori was cultured with 100 μM of anthocyanin in Mueller-Hinton broth/10% FBS for 3 days and proteins assessed by Western blot. (A), secreted CagA and VacA in culture media. (B), secreted H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. (C) , intracellular CagA and VacA. (D) , intracellular H. pylori proteins reacting with rabbit anti- H. pylori polyclonal antibody. Representative image from five independent experiments.

    Article Snippet: Membranes were blocked with 5% skim milk for 30 minutes and then incubated with mouse anti-CagA monoclonal antibody (Santa Cruz Biotechnology, CA, USA), rabbit anti-VacA polyclonal antibody (Santa Cruz Biotechnology) or rabbit anti-Helicobacter pylori polyclonal antibody (this study).

    Techniques: Cell Culture, Western Blot

    Ubiquitination of total proteins and proteasome activity in AGS cells co-cultured with H. pylori . (A) Representative immunoblot of total ubiquitinated protein from AGS cells infected with H. pylori (MOI = 300) for 8 h, in presence or absence of 2.5 μM Lactacystin. Tubulin is shown as loading control and CagA as infection control. (B) Quantification of proteasome activity in AGS cells infected with H. pylori wild type and its isogenic mutants cagA, vacA and, in presence of 2.5 μM Lactacystin for 8 h. Asterisk represents significant difference ( p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: KCTD5 and Ubiquitin Proteasome Signaling Are Required for Helicobacter pylori Adherence

    doi: 10.3389/fcimb.2017.00450

    Figure Lengend Snippet: Ubiquitination of total proteins and proteasome activity in AGS cells co-cultured with H. pylori . (A) Representative immunoblot of total ubiquitinated protein from AGS cells infected with H. pylori (MOI = 300) for 8 h, in presence or absence of 2.5 μM Lactacystin. Tubulin is shown as loading control and CagA as infection control. (B) Quantification of proteasome activity in AGS cells infected with H. pylori wild type and its isogenic mutants cagA, vacA and, in presence of 2.5 μM Lactacystin for 8 h. Asterisk represents significant difference ( p

    Article Snippet: Anti-Tubulin mouse monoclonal (Catalog number: T5168, Sigma-Aldrich, St Louis, Missouri, USA) and anti-CagA mouse monoclonal antibodies (Catalog number: sc-28368, Santa Cruz Biotechnology, Dallas, Texas, USA) were used as loading and H. pylori -infection controls, respectively.

    Techniques: Activity Assay, Cell Culture, Infection