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Santa Cruz Biotechnology anti caga
<t>CagA</t> + H. pylori -induced <t>HIF-1α</t> stabilization is regulated by prolyl hydroxylase (PHD) activity (A) AGS cells were pretreated with or without 10 μM MG-132 for 1 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with antibodies specific for hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α. (B) After infection with H. pylori for 6 h, immunoblotting was performed using the indicated antibodies. (C) AGS cells were pretreated with or without 1mM DMOG for 2 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with anti-HIF-1α antibody. Densitometric ratios between HIF-1α and β-actin immunoblotting are shown at the bottom. (D) After treatment with the indicated inhibitors prior to CagA + H. pylori infection, HIF-1α protein levels were detected by immunoblotting. Data are represented as mean ± SD (n = 3).
Anti Caga, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA"

Article Title: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA

Journal: Oncotarget

doi: 10.18632/oncotarget.18695

CagA + H. pylori -induced HIF-1α stabilization is regulated by prolyl hydroxylase (PHD) activity (A) AGS cells were pretreated with or without 10 μM MG-132 for 1 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with antibodies specific for hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α. (B) After infection with H. pylori for 6 h, immunoblotting was performed using the indicated antibodies. (C) AGS cells were pretreated with or without 1mM DMOG for 2 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with anti-HIF-1α antibody. Densitometric ratios between HIF-1α and β-actin immunoblotting are shown at the bottom. (D) After treatment with the indicated inhibitors prior to CagA + H. pylori infection, HIF-1α protein levels were detected by immunoblotting. Data are represented as mean ± SD (n = 3).
Figure Legend Snippet: CagA + H. pylori -induced HIF-1α stabilization is regulated by prolyl hydroxylase (PHD) activity (A) AGS cells were pretreated with or without 10 μM MG-132 for 1 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with antibodies specific for hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α. (B) After infection with H. pylori for 6 h, immunoblotting was performed using the indicated antibodies. (C) AGS cells were pretreated with or without 1mM DMOG for 2 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with anti-HIF-1α antibody. Densitometric ratios between HIF-1α and β-actin immunoblotting are shown at the bottom. (D) After treatment with the indicated inhibitors prior to CagA + H. pylori infection, HIF-1α protein levels were detected by immunoblotting. Data are represented as mean ± SD (n = 3).

Techniques Used: Activity Assay, Infection

Proposed model depicting the regulation of HIF-1α by downregulated SIRT3-mediated ROS production in H. pylori -infected gastric cells Infection with H. pylori CagA + induces SIRT3 protein degradation by proteasome activity. Increased ROS generation from the downregulated SIRT3 activity mediates the activation of HIF-1α, and this, in turn, induces the expression of HIF-1α target genes, which leads to gastric cancer proliferation.
Figure Legend Snippet: Proposed model depicting the regulation of HIF-1α by downregulated SIRT3-mediated ROS production in H. pylori -infected gastric cells Infection with H. pylori CagA + induces SIRT3 protein degradation by proteasome activity. Increased ROS generation from the downregulated SIRT3 activity mediates the activation of HIF-1α, and this, in turn, induces the expression of HIF-1α target genes, which leads to gastric cancer proliferation.

Techniques Used: Infection, Activity Assay, Activation Assay, Expressing

H. pylori CagA increases HIF-1α activity and induces expression of HIF-1α target genes (A) AGS cells were infected with H. pylori strains for 6 h and the CagA protein level was analyzed by immunoblotting. (B) Lysates from AGS and MKN28 cells infected with H. pylori strains were immunoblotted with anti-HIF-1α, and then, the membrane was stripped and analyzed with anti-β-actin to standardize gel loading. (C) MKN28 cells were infected with H. pylori strains for 6 h, and then, cytoplasmic and nuclear fractions were separated. The nuclear proteins were analyzed by immunoblotting with anti-HIF-1α. To verify complete separation of the cytosolic and nuclear fractions, cytosolic and nuclear extracts were immunoblotted for aldolase A and lamin B. (D) MKN28 cells were co-transfected with HRE-Luc together with TK-renilla for 24 h and then infected with H. pylori strains for 12 h. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (E) MKN28 cells were infected with H. pylori for 12 h, and the fold change in HIF-1α target gene levels was measured by real-time PCR using specific primers for Vegf-a , Pdk-1, Ldha, and Hif-1α . Data are represented as mean ± SD (n = 3).
Figure Legend Snippet: H. pylori CagA increases HIF-1α activity and induces expression of HIF-1α target genes (A) AGS cells were infected with H. pylori strains for 6 h and the CagA protein level was analyzed by immunoblotting. (B) Lysates from AGS and MKN28 cells infected with H. pylori strains were immunoblotted with anti-HIF-1α, and then, the membrane was stripped and analyzed with anti-β-actin to standardize gel loading. (C) MKN28 cells were infected with H. pylori strains for 6 h, and then, cytoplasmic and nuclear fractions were separated. The nuclear proteins were analyzed by immunoblotting with anti-HIF-1α. To verify complete separation of the cytosolic and nuclear fractions, cytosolic and nuclear extracts were immunoblotted for aldolase A and lamin B. (D) MKN28 cells were co-transfected with HRE-Luc together with TK-renilla for 24 h and then infected with H. pylori strains for 12 h. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (E) MKN28 cells were infected with H. pylori for 12 h, and the fold change in HIF-1α target gene levels was measured by real-time PCR using specific primers for Vegf-a , Pdk-1, Ldha, and Hif-1α . Data are represented as mean ± SD (n = 3).

Techniques Used: Activity Assay, Expressing, Infection, Transfection, Luciferase, Real-time Polymerase Chain Reaction

H. pylori CagA protein localizes to mitochondria and is involved in ROS production (A) Cytoplasmic (Cyt) and mitochondrial (Mito) proteins were fractionated from H. pylori -infected AGS cells and analyzed by immunoblotting (left panel) and immunofluorescence (right panel) with anti-CagA antibody. In immunofluorescence microscopy, CagA was stained in green, mitochondria in red, and DNA in blue with 4’,6-diamidino-2-phenylindole (magnification x100). (B) AGS cells were infected with H. pylori for 6 h and stained with Mitosox, followed by immunofluorescence (left panel) and FACS analysis (right panel). (C) AGS cells were pretreated with antioxidants (NAC, catalase, allopurinol, and DESF) prior to H. pylori infection and extracts were immunoblotted with HIF-1α antibody. (D) AGS cells were pretreated with DPI or myxothiazol and then the extracts were immunoblotted (left panel). AGS cell were co-transfected with HRE-Luc and TK-renilla constructs for 24 h and treated with the inhibitors DPI or myxothiazol for 1 h. Cells were then infected with CagA + H. pylori strains for 6 h, and luciferase reporter activity was measured. Data are represented as mean ± SD (n = 3).
Figure Legend Snippet: H. pylori CagA protein localizes to mitochondria and is involved in ROS production (A) Cytoplasmic (Cyt) and mitochondrial (Mito) proteins were fractionated from H. pylori -infected AGS cells and analyzed by immunoblotting (left panel) and immunofluorescence (right panel) with anti-CagA antibody. In immunofluorescence microscopy, CagA was stained in green, mitochondria in red, and DNA in blue with 4’,6-diamidino-2-phenylindole (magnification x100). (B) AGS cells were infected with H. pylori for 6 h and stained with Mitosox, followed by immunofluorescence (left panel) and FACS analysis (right panel). (C) AGS cells were pretreated with antioxidants (NAC, catalase, allopurinol, and DESF) prior to H. pylori infection and extracts were immunoblotted with HIF-1α antibody. (D) AGS cells were pretreated with DPI or myxothiazol and then the extracts were immunoblotted (left panel). AGS cell were co-transfected with HRE-Luc and TK-renilla constructs for 24 h and treated with the inhibitors DPI or myxothiazol for 1 h. Cells were then infected with CagA + H. pylori strains for 6 h, and luciferase reporter activity was measured. Data are represented as mean ± SD (n = 3).

Techniques Used: Infection, Immunofluorescence, Microscopy, Staining, FACS, Transfection, Construct, Luciferase, Activity Assay

2) Product Images from "Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells"

Article Title: Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells

Journal: International Journal of Medical Sciences

doi:

Detection of CagA and VacA protein by Western Blot. a: CagA protein (arrow) b: VacA protein (arrow). Lane 1: Marker; Lane 2: Hp 11638M; Lane 3: Hp11638.
Figure Legend Snippet: Detection of CagA and VacA protein by Western Blot. a: CagA protein (arrow) b: VacA protein (arrow). Lane 1: Marker; Lane 2: Hp 11638M; Lane 3: Hp11638.

Techniques Used: Western Blot, Marker

3) Product Images from "Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway"

Article Title: Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0962-5

a Expression of E-cadherin (an epithelial marker) and N-cadherin (a mesenchymal marker) in AGS cells transfected with YAP cDNA plasmid. b After AGS cells were infected with H. pylori strains 7.13 or PMSS1 and their cagA − mutants, Expression of E-cadherin (red) were visualized using Immunofluorescence. c Immunofluorescence was performed for E-cadherin levels. d mRNA levels of YAP downstream genes CTGF, CYR61 and E-cadherin (an epithelial marker) in AGS cells infected with H. pylori strains alone or in combination with VP treatment. e Expression of YAP and mesenchymal markers Slug and N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with VP. f Expression of YAP, epithelial markers E-cadherin and mesenchymal markers N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with YAP siRNA. Data for gene expression are mean ± SEM of 3 independent experiments
Figure Legend Snippet: a Expression of E-cadherin (an epithelial marker) and N-cadherin (a mesenchymal marker) in AGS cells transfected with YAP cDNA plasmid. b After AGS cells were infected with H. pylori strains 7.13 or PMSS1 and their cagA − mutants, Expression of E-cadherin (red) were visualized using Immunofluorescence. c Immunofluorescence was performed for E-cadherin levels. d mRNA levels of YAP downstream genes CTGF, CYR61 and E-cadherin (an epithelial marker) in AGS cells infected with H. pylori strains alone or in combination with VP treatment. e Expression of YAP and mesenchymal markers Slug and N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with VP. f Expression of YAP, epithelial markers E-cadherin and mesenchymal markers N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with YAP siRNA. Data for gene expression are mean ± SEM of 3 independent experiments

Techniques Used: Expressing, Marker, Transfection, Plasmid Preparation, Infection, Immunofluorescence

4) Product Images from "Helicobacter pylori-derived extracellular vesicles increased in the gastric juices of gastric adenocarcinoma patients and induced inflammation mainly via specific targeting of gastric epithelial cells"

Article Title: Helicobacter pylori-derived extracellular vesicles increased in the gastric juices of gastric adenocarcinoma patients and induced inflammation mainly via specific targeting of gastric epithelial cells

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2017.47

Characterization of purified H. pylori -derived extracellular vesicles (EVs) and induction of IL-8 with EVs in vitro . ( a ) Transmission electron microscopy (TEM) images. ( b ) Size distribution of EVs determined using dynamic light scattering (DLS). ( c ) Confirmation of CagA and VacA in cell extracts and EVs by western blotting. Hp, HpΔ CagA and HpEV denote cell extracts from wild-type H. pylori strain HP99 (Hp), isogenic H. pylori with CagA gene deletion (HpΔ CagA ), and EVs from H. pylori strain HP99 (EV), respectively. ( d ) Induction of IL-8 in human gastric adenocarcinoma cell line AGS by IL-1β (positive control) and EVs (* P
Figure Legend Snippet: Characterization of purified H. pylori -derived extracellular vesicles (EVs) and induction of IL-8 with EVs in vitro . ( a ) Transmission electron microscopy (TEM) images. ( b ) Size distribution of EVs determined using dynamic light scattering (DLS). ( c ) Confirmation of CagA and VacA in cell extracts and EVs by western blotting. Hp, HpΔ CagA and HpEV denote cell extracts from wild-type H. pylori strain HP99 (Hp), isogenic H. pylori with CagA gene deletion (HpΔ CagA ), and EVs from H. pylori strain HP99 (EV), respectively. ( d ) Induction of IL-8 in human gastric adenocarcinoma cell line AGS by IL-1β (positive control) and EVs (* P

Techniques Used: Purification, Derivative Assay, In Vitro, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot, Positive Control

5) Product Images from "Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway"

Article Title: Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0962-5

a Expression of E-cadherin (an epithelial marker) and N-cadherin (a mesenchymal marker) in AGS cells transfected with YAP cDNA plasmid. b After AGS cells were infected with H. pylori strains 7.13 or PMSS1 and their cagA − mutants, Expression of E-cadherin (red) were visualized using Immunofluorescence. c Immunofluorescence was performed for E-cadherin levels. d mRNA levels of YAP downstream genes CTGF, CYR61 and E-cadherin (an epithelial marker) in AGS cells infected with H. pylori strains alone or in combination with VP treatment. e Expression of YAP and mesenchymal markers Slug and N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with VP. f Expression of YAP, epithelial markers E-cadherin and mesenchymal markers N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with YAP siRNA. Data for gene expression are mean ± SEM of 3 independent experiments
Figure Legend Snippet: a Expression of E-cadherin (an epithelial marker) and N-cadherin (a mesenchymal marker) in AGS cells transfected with YAP cDNA plasmid. b After AGS cells were infected with H. pylori strains 7.13 or PMSS1 and their cagA − mutants, Expression of E-cadherin (red) were visualized using Immunofluorescence. c Immunofluorescence was performed for E-cadherin levels. d mRNA levels of YAP downstream genes CTGF, CYR61 and E-cadherin (an epithelial marker) in AGS cells infected with H. pylori strains alone or in combination with VP treatment. e Expression of YAP and mesenchymal markers Slug and N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with VP. f Expression of YAP, epithelial markers E-cadherin and mesenchymal markers N-cadherin in AGS cells treated with CagA + H. pylori PMSS1 or 7.13 alone or in combination with YAP siRNA. Data for gene expression are mean ± SEM of 3 independent experiments

Techniques Used: Expressing, Marker, Transfection, Plasmid Preparation, Infection, Immunofluorescence

6) Product Images from "VacA promotes CagA accumulation in gastric epithelial cells during Helicobacter pylori infection"

Article Title: VacA promotes CagA accumulation in gastric epithelial cells during Helicobacter pylori infection

Journal: Scientific Reports

doi: 10.1038/s41598-018-37095-4

VacA disrupts autophagy but does not impair proteasome degradation. ( A ) AGS cells were infected with a CagA+ vacA isogenic mutant strain and co-cultured in the presence or absence of VacA− or VacA+ CCMS for 19 hours using a gentamycin protection assay. MG132 (5 μM) was added during the 14-hour low-dose gentamycin incubation. DMSO was used as a vehicle control. Ubiquitin protein levels were measured by Western blotting using β-actin as loading control. Graph shows fold change of ubiquitin normalized to β-actin relative to vehicle control (mean + SEM; n = 5). ( B ) Ub G76V -GFP HeLa cells were treated with 10 μM of MG132 or Lactacystin for 4 and 8 hours respectively, or VacA+ or VacA− CCMS for 32 hours. Ub G76V -GFP protein levels were measured by Western blotting using β-actin as loading control (mean + SEM; n = 3). Relevant gel bands were cropped from the original blots. Dotted line indicates slicing of two regions together from the same blot. Statistical analysis was performed using Student’s t-test.
Figure Legend Snippet: VacA disrupts autophagy but does not impair proteasome degradation. ( A ) AGS cells were infected with a CagA+ vacA isogenic mutant strain and co-cultured in the presence or absence of VacA− or VacA+ CCMS for 19 hours using a gentamycin protection assay. MG132 (5 μM) was added during the 14-hour low-dose gentamycin incubation. DMSO was used as a vehicle control. Ubiquitin protein levels were measured by Western blotting using β-actin as loading control. Graph shows fold change of ubiquitin normalized to β-actin relative to vehicle control (mean + SEM; n = 5). ( B ) Ub G76V -GFP HeLa cells were treated with 10 μM of MG132 or Lactacystin for 4 and 8 hours respectively, or VacA+ or VacA− CCMS for 32 hours. Ub G76V -GFP protein levels were measured by Western blotting using β-actin as loading control (mean + SEM; n = 3). Relevant gel bands were cropped from the original blots. Dotted line indicates slicing of two regions together from the same blot. Statistical analysis was performed using Student’s t-test.

Techniques Used: Infection, Mutagenesis, Cell Culture, Incubation, Western Blot

VacA promotes the accumulation of phosphorylated CagA. AGS cells were infected with a CagA+ vacA − isogenic mutant strain in the presence of (A) VacA− or VacA+ CCMS and ( B ) untreated or treated with purified VacA toxin for 20 hours using a gentamycin protection assay. Phosphorylated CagA (pTyr) and total CagA levels were measured by Western blotting using β-actin as a loading control. ( C ) AGS cells were infected with a CagA+ vacA − isogenic mutant strain in the presence of VacA− or VacA+ CCMS for 19 hours using a gentamycin protection assay and incubated with DMSO or MG132 (5 μM). Phosphorylated CagA levels were measured by Western blotting using β-actin as loading control. Graphs show fold change of pTyr or CagA normalized to β-actin (mean + SEM; n = 3). Statistical analysis was performed using Student’s t-test.
Figure Legend Snippet: VacA promotes the accumulation of phosphorylated CagA. AGS cells were infected with a CagA+ vacA − isogenic mutant strain in the presence of (A) VacA− or VacA+ CCMS and ( B ) untreated or treated with purified VacA toxin for 20 hours using a gentamycin protection assay. Phosphorylated CagA (pTyr) and total CagA levels were measured by Western blotting using β-actin as a loading control. ( C ) AGS cells were infected with a CagA+ vacA − isogenic mutant strain in the presence of VacA− or VacA+ CCMS for 19 hours using a gentamycin protection assay and incubated with DMSO or MG132 (5 μM). Phosphorylated CagA levels were measured by Western blotting using β-actin as loading control. Graphs show fold change of pTyr or CagA normalized to β-actin (mean + SEM; n = 3). Statistical analysis was performed using Student’s t-test.

Techniques Used: Infection, Mutagenesis, Purification, Western Blot, Incubation

Related Articles

Western Blot:

Article Title: VacA promotes CagA accumulation in gastric epithelial cells during Helicobacter pylori infection
Article Snippet: .. Antibodies The following antibodies were used for Western blotting: anti-β-actin (A4700; 1:5000; Sigma-Aldrich, Oakville, Canada), anti-LC3B (NB600-1384; 1:1000 for WB; 1:200 for IF; Novus Biologicals; Oakville, Canada), anti-p62 (610832; 1:1000; BD Transduction; Mississauga, Canada), anti-pTyr (4G10; 1:500; EMD Millipore, Etobicoke, Canada), anti-CagA (b-300; 1:500; Santa Cruz, Dallas, TX), anti-Ubiquitin (P4D1; 1:500; Santa Cruz, Dallas, TX), anti-Atg12 (2010; 1:500; Cell Signaling, Whitby, Canada) goat anti-mouse IgG (115-035-003; 1:2500; Jackson ImmunoResearch, West Grove, PA), and goat anti-rabbit IgG (115-035-144; 1:2500; Jackson ImmunoResearch, West Grove, PA). .. Pharmacological inhibitors MG132 (5 μM; Sigma-Aldrich, Oakville, Canada) was dissolved in DMSO and Lactacystin (10 μM; Enzo Life Sciences, Farmingdale, NY) was dissolved in milli-Q water.

Article Title: Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway
Article Snippet: .. Antibodies and their sources were as follows: YAP inhibitor verteporfin from Sigma-Aldrich (St. Louis, MO, USA) for western blot assay, Anti-GAPDH (#2118), Anti-YAP (#4912), Anti-Phospho-YAP Ser127 (#4911), and Anti-Slug (#9585), from Cell Signaling Technology (Beverly, MA, USA); Anti-TAZ (HPA007415) from Sigma (St. Louis, MO, USA) (Anti-E-cadherin (#610405), Anti-N-cadherin (#610921) from BD Biosciences (San Jose, CA USA); Anti-CagA (sc-28,368) and Anti-phospho-tyrosine (sc-7020) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti- H. pylori urease B (ab127916) from Abcam (Cambridge, MA, USA). .. For immunohistochemistry assay, Anti-YAP (#4912) from Cell Signaling Technology, Anti-TAZ (HPA007415) from Sigma, Anti-E-cadherin (#610405) from BD Biosciences For immunofluorescence assay, Anti-YAP (#4912) from Cell Signaling Technology, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies from Invitrogen (Thermo Fisher Scientific, Suwanee, GA, USA).

Article Title: Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway
Article Snippet: .. Antibodies, siRNA and plasmids Antibodies and their sources were as follows: YAP inhibitor verteporfin from Sigma-Aldrich (St. Louis, MO, USA) for western blot assay, Anti-GAPDH (#2118), Anti-YAP (#4912), Anti-Phospho-YAP Ser127 (#4911), and Anti-Slug (#9585), from Cell Signaling Technology (Beverly, MA, USA); Anti-TAZ (HPA007415) from Sigma (St. Louis, MO, USA) (Anti-E-cadherin (#610405), Anti-N-cadherin (#610921) from BD Biosciences (San Jose, CA USA); Anti-CagA (sc-28,368) and Anti-phospho-tyrosine (sc-7020) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-H. pylori urease B (ab127916) from Abcam (Cambridge, MA, USA). .. For immunohistochemistry assay, Anti-YAP (#4912) from Cell Signaling Technology, Anti-TAZ (HPA007415) from Sigma, Anti-E-cadherin (#610405) from BD Biosciences For immunofluorescence assay, Anti-YAP (#4912) from Cell Signaling Technology, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies from Invitrogen (Thermo Fisher Scientific, Suwanee, GA, USA).

Chloramphenicol Acetyltransferase Assay:

Article Title: Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells
Article Snippet: .. Reagents F12 culture medium (Hangzhou Jino Biology Co., Ltd. China), fetal bovine serum (FBS), ampicillin, penicillin and streptomycin (Shanghai Bio-engineering Co., Ltd. China), brain heart infusion agar and liquid medium (OXOID Co., Ltd. UK), LB medium (Beijing Solarbio Co., Ltd. China), gas mixture (5% O2 , 85% N2 , 10% CO2 ) (Shanghai Shenkai Gas Co., Ltd. China), anti-CagA and anti-VacA polyclonal antibodies (Santa Cruz, USA), AP conjugated secondary antibody, CellTiter-Glo luminescent cell viability assay kit (Cat.#G7570, Promega Co. USA), Dihydrorhdamine-123 (DHR-123) and oxidized cytochrome c (Sigma, USA) and AGS mtDNA extraction kit GenMed Scientifics Co., Ltd USA) were used in the present study. ..

other:

Article Title: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA
Article Snippet: Antibodies and chemicals Anti-HIF-1α, anti-CagA, anti-PHD1, anti-PHD3, anti-Aldolase A, anti-Lamin B, and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cell Viability Assay:

Article Title: Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells
Article Snippet: .. Reagents F12 culture medium (Hangzhou Jino Biology Co., Ltd. China), fetal bovine serum (FBS), ampicillin, penicillin and streptomycin (Shanghai Bio-engineering Co., Ltd. China), brain heart infusion agar and liquid medium (OXOID Co., Ltd. UK), LB medium (Beijing Solarbio Co., Ltd. China), gas mixture (5% O2 , 85% N2 , 10% CO2 ) (Shanghai Shenkai Gas Co., Ltd. China), anti-CagA and anti-VacA polyclonal antibodies (Santa Cruz, USA), AP conjugated secondary antibody, CellTiter-Glo luminescent cell viability assay kit (Cat.#G7570, Promega Co. USA), Dihydrorhdamine-123 (DHR-123) and oxidized cytochrome c (Sigma, USA) and AGS mtDNA extraction kit GenMed Scientifics Co., Ltd USA) were used in the present study. ..

Incubation:

Article Title: Helicobacter pylori-derived extracellular vesicles increased in the gastric juices of gastric adenocarcinoma patients and induced inflammation mainly via specific targeting of gastric epithelial cells
Article Snippet: .. The membranes were blocked with 5% skim milk and incubated with anti-CagA (sc-25766) and anti-VacA (sc-25790) antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and secondary antibodies before development. .. Analyses of in vitro cytokine production

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  • 88
    Santa Cruz Biotechnology anti caga
    <t>CagA</t> + H. pylori -induced <t>HIF-1α</t> stabilization is regulated by prolyl hydroxylase (PHD) activity (A) AGS cells were pretreated with or without 10 μM MG-132 for 1 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with antibodies specific for hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α. (B) After infection with H. pylori for 6 h, immunoblotting was performed using the indicated antibodies. (C) AGS cells were pretreated with or without 1mM DMOG for 2 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with anti-HIF-1α antibody. Densitometric ratios between HIF-1α and β-actin immunoblotting are shown at the bottom. (D) After treatment with the indicated inhibitors prior to CagA + H. pylori infection, HIF-1α protein levels were detected by immunoblotting. Data are represented as mean ± SD (n = 3).
    Anti Caga, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti caga/product/Santa Cruz Biotechnology
    Average 88 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
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    Santa Cruz Biotechnology rabbit igg anti caga polyclonal antibody b 300
    Expression, delivery, and phosphorylation of <t>CagA.</t> (A) Western blot analysis of bacterial lysates from the six indicated Korean strains was conducted using a monoclonal anti-CagA antibody (top) or the <t>polyclonal</t> anti-CagA antibody b-300 (bottom). (B)
    Rabbit Igg Anti Caga Polyclonal Antibody B 300, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit igg anti caga polyclonal antibody b 300/product/Santa Cruz Biotechnology
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    Santa Cruz Biotechnology polyclonal anti caga antibody b 300
    Analysis of the <t>CagA</t> protein patterns by immunoblot analysis. (A) Lysates of H . pylori isolated from the tissue specimens of various gastric patients were prepared as described in the Materials and Methods. Aliquots of protein (20 μg) were separated by SDS-gel electrophoresis and CagA was detected by immunoblotting using <t>polyclonal</t> anti-CagA antibody b-300 (Santa Cruz). The H . pylori 60190 and 26695 cagA knockout mutant (26695ΔA) were included as a positive and negative control, respectively, for CagA expression. The numbers at the top identify the various H . pylori colonies, while those on the left indicate the molecular weight markers in kDa. It can be seen that there are four types of CagA fragmentation patterns. (B) The percentages of ABD subtypes based on the CagA protein fragmentation patterns across the 96 H . pylori cagA ABD colonies that were analyzed and clearly ABD-2 is the predominant type.
    Polyclonal Anti Caga Antibody B 300, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti caga antibody b 300/product/Santa Cruz Biotechnology
    Average 90 stars, based on 2 article reviews
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    CagA + H. pylori -induced HIF-1α stabilization is regulated by prolyl hydroxylase (PHD) activity (A) AGS cells were pretreated with or without 10 μM MG-132 for 1 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with antibodies specific for hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α. (B) After infection with H. pylori for 6 h, immunoblotting was performed using the indicated antibodies. (C) AGS cells were pretreated with or without 1mM DMOG for 2 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with anti-HIF-1α antibody. Densitometric ratios between HIF-1α and β-actin immunoblotting are shown at the bottom. (D) After treatment with the indicated inhibitors prior to CagA + H. pylori infection, HIF-1α protein levels were detected by immunoblotting. Data are represented as mean ± SD (n = 3).

    Journal: Oncotarget

    Article Title: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA

    doi: 10.18632/oncotarget.18695

    Figure Lengend Snippet: CagA + H. pylori -induced HIF-1α stabilization is regulated by prolyl hydroxylase (PHD) activity (A) AGS cells were pretreated with or without 10 μM MG-132 for 1 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with antibodies specific for hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α. (B) After infection with H. pylori for 6 h, immunoblotting was performed using the indicated antibodies. (C) AGS cells were pretreated with or without 1mM DMOG for 2 h and infected with H. pylori for 6 h. Cell lysates were immunoblotted with anti-HIF-1α antibody. Densitometric ratios between HIF-1α and β-actin immunoblotting are shown at the bottom. (D) After treatment with the indicated inhibitors prior to CagA + H. pylori infection, HIF-1α protein levels were detected by immunoblotting. Data are represented as mean ± SD (n = 3).

    Article Snippet: Antibodies and chemicals Anti-HIF-1α, anti-CagA, anti-PHD1, anti-PHD3, anti-Aldolase A, anti-Lamin B, and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Activity Assay, Infection

    Proposed model depicting the regulation of HIF-1α by downregulated SIRT3-mediated ROS production in H. pylori -infected gastric cells Infection with H. pylori CagA + induces SIRT3 protein degradation by proteasome activity. Increased ROS generation from the downregulated SIRT3 activity mediates the activation of HIF-1α, and this, in turn, induces the expression of HIF-1α target genes, which leads to gastric cancer proliferation.

    Journal: Oncotarget

    Article Title: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA

    doi: 10.18632/oncotarget.18695

    Figure Lengend Snippet: Proposed model depicting the regulation of HIF-1α by downregulated SIRT3-mediated ROS production in H. pylori -infected gastric cells Infection with H. pylori CagA + induces SIRT3 protein degradation by proteasome activity. Increased ROS generation from the downregulated SIRT3 activity mediates the activation of HIF-1α, and this, in turn, induces the expression of HIF-1α target genes, which leads to gastric cancer proliferation.

    Article Snippet: Antibodies and chemicals Anti-HIF-1α, anti-CagA, anti-PHD1, anti-PHD3, anti-Aldolase A, anti-Lamin B, and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Infection, Activity Assay, Activation Assay, Expressing

    H. pylori CagA increases HIF-1α activity and induces expression of HIF-1α target genes (A) AGS cells were infected with H. pylori strains for 6 h and the CagA protein level was analyzed by immunoblotting. (B) Lysates from AGS and MKN28 cells infected with H. pylori strains were immunoblotted with anti-HIF-1α, and then, the membrane was stripped and analyzed with anti-β-actin to standardize gel loading. (C) MKN28 cells were infected with H. pylori strains for 6 h, and then, cytoplasmic and nuclear fractions were separated. The nuclear proteins were analyzed by immunoblotting with anti-HIF-1α. To verify complete separation of the cytosolic and nuclear fractions, cytosolic and nuclear extracts were immunoblotted for aldolase A and lamin B. (D) MKN28 cells were co-transfected with HRE-Luc together with TK-renilla for 24 h and then infected with H. pylori strains for 12 h. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (E) MKN28 cells were infected with H. pylori for 12 h, and the fold change in HIF-1α target gene levels was measured by real-time PCR using specific primers for Vegf-a , Pdk-1, Ldha, and Hif-1α . Data are represented as mean ± SD (n = 3).

    Journal: Oncotarget

    Article Title: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA

    doi: 10.18632/oncotarget.18695

    Figure Lengend Snippet: H. pylori CagA increases HIF-1α activity and induces expression of HIF-1α target genes (A) AGS cells were infected with H. pylori strains for 6 h and the CagA protein level was analyzed by immunoblotting. (B) Lysates from AGS and MKN28 cells infected with H. pylori strains were immunoblotted with anti-HIF-1α, and then, the membrane was stripped and analyzed with anti-β-actin to standardize gel loading. (C) MKN28 cells were infected with H. pylori strains for 6 h, and then, cytoplasmic and nuclear fractions were separated. The nuclear proteins were analyzed by immunoblotting with anti-HIF-1α. To verify complete separation of the cytosolic and nuclear fractions, cytosolic and nuclear extracts were immunoblotted for aldolase A and lamin B. (D) MKN28 cells were co-transfected with HRE-Luc together with TK-renilla for 24 h and then infected with H. pylori strains for 12 h. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (E) MKN28 cells were infected with H. pylori for 12 h, and the fold change in HIF-1α target gene levels was measured by real-time PCR using specific primers for Vegf-a , Pdk-1, Ldha, and Hif-1α . Data are represented as mean ± SD (n = 3).

    Article Snippet: Antibodies and chemicals Anti-HIF-1α, anti-CagA, anti-PHD1, anti-PHD3, anti-Aldolase A, anti-Lamin B, and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Activity Assay, Expressing, Infection, Transfection, Luciferase, Real-time Polymerase Chain Reaction

    H. pylori CagA protein localizes to mitochondria and is involved in ROS production (A) Cytoplasmic (Cyt) and mitochondrial (Mito) proteins were fractionated from H. pylori -infected AGS cells and analyzed by immunoblotting (left panel) and immunofluorescence (right panel) with anti-CagA antibody. In immunofluorescence microscopy, CagA was stained in green, mitochondria in red, and DNA in blue with 4’,6-diamidino-2-phenylindole (magnification x100). (B) AGS cells were infected with H. pylori for 6 h and stained with Mitosox, followed by immunofluorescence (left panel) and FACS analysis (right panel). (C) AGS cells were pretreated with antioxidants (NAC, catalase, allopurinol, and DESF) prior to H. pylori infection and extracts were immunoblotted with HIF-1α antibody. (D) AGS cells were pretreated with DPI or myxothiazol and then the extracts were immunoblotted (left panel). AGS cell were co-transfected with HRE-Luc and TK-renilla constructs for 24 h and treated with the inhibitors DPI or myxothiazol for 1 h. Cells were then infected with CagA + H. pylori strains for 6 h, and luciferase reporter activity was measured. Data are represented as mean ± SD (n = 3).

    Journal: Oncotarget

    Article Title: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA

    doi: 10.18632/oncotarget.18695

    Figure Lengend Snippet: H. pylori CagA protein localizes to mitochondria and is involved in ROS production (A) Cytoplasmic (Cyt) and mitochondrial (Mito) proteins were fractionated from H. pylori -infected AGS cells and analyzed by immunoblotting (left panel) and immunofluorescence (right panel) with anti-CagA antibody. In immunofluorescence microscopy, CagA was stained in green, mitochondria in red, and DNA in blue with 4’,6-diamidino-2-phenylindole (magnification x100). (B) AGS cells were infected with H. pylori for 6 h and stained with Mitosox, followed by immunofluorescence (left panel) and FACS analysis (right panel). (C) AGS cells were pretreated with antioxidants (NAC, catalase, allopurinol, and DESF) prior to H. pylori infection and extracts were immunoblotted with HIF-1α antibody. (D) AGS cells were pretreated with DPI or myxothiazol and then the extracts were immunoblotted (left panel). AGS cell were co-transfected with HRE-Luc and TK-renilla constructs for 24 h and treated with the inhibitors DPI or myxothiazol for 1 h. Cells were then infected with CagA + H. pylori strains for 6 h, and luciferase reporter activity was measured. Data are represented as mean ± SD (n = 3).

    Article Snippet: Antibodies and chemicals Anti-HIF-1α, anti-CagA, anti-PHD1, anti-PHD3, anti-Aldolase A, anti-Lamin B, and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Infection, Immunofluorescence, Microscopy, Staining, FACS, Transfection, Construct, Luciferase, Activity Assay

    Variation in the CagA amino acid sequence of East Asian-type and Western-type CagA. The target α-EAS sequences “AINRKIDRINKIASAGKG” in EPIYA segment D, based on the sequences of Japanese strains, are shown in frame. A sequence analysis revealed that the typical target sequences in Bhutanese strains was “(T/K)IN(G/R)KID(Q/R)(L/I)N(R/K)(T/I)ASA(G/N)KG,” where (X/Y) means X and Y as the two major amino acids. The sequences of the EPIYA-D segments in Bhutanese strains were highly variable compared to strains deposited in GenBank. In contrast, the East Asian-type and Western-type sequences of EPIYA-C segments in Bhutanese strains were largely identical, similar to the typical sequences of EPIYA-C segments deposited in GenBank. Reference strains used in Fig. 1A (strain name [accession number]) were 103a (AB110966.1), 105a (AB110967.1), 106a (AB110968.1), 108a (AB110969.1), 113b (AB110970.1), 120a (AB110971.1), 122b (AB110972.1), 125b (AB110973.1), 128a (AB110974.1), FJT77 (KF028580.1), 04-518 (AB267252.1), 03-166 (AB267253.1), 04-264 (AB267254.1), THP1477 (AB116744.1), 04-334 (AB267249.1), 03-292 (AB267250.1), 04-366 (AB267251.1), THP1260 (AB116742.1), M3 (AB116740.1), THP463 (AB116735.1), Korea23 (AB057044.1), Korea 12 (AB057043.1), K69 (FJ458129.1), Korea2-3 (AB057040.1), k266 (FJ458163.1), K265 (FJ458162.1), K264 (FJ458161.1), K261 (FJ458158.1), K260 (FJ458157.1), K259 (FJ458156.1). Reference strains used in Fig. 1C (strain name [accession number]) were India41 (AF222807.1), India99 (AF222809.1), OSC40A (EU089774.1), OSC42B (EU089775.1), PCR-156i (EU368669.1), PCR218vi (EU089766.1). RIGLD-OC149 (JX428784.1), SAN53 (EU089771.1), PD682 (EF450167.1), PD636 (EF450165.1), PD308 (EF450162.1), PD488 (EF450161.1), PD537 (EF450160.1), PD501 (EF450159.1), PD351 (EF450158.1), PD348 (EF450157.1), PD6481K (EF450153.1), 216G (GQ899171.1), 1407 (GU143415.1), HPI-14 (FJ849792.1), HPI-13 (FJ849791.1), HPI-11 (FJ849789.1), USA2791 (AB057099.1), Kazak3 (AB057098.1), USA35 (AB057095.1), Italy329 (AB057094.1), Arizona2 (AB057075.1), Arizona1 (AB057074.1).

    Journal: Scientific Reports

    Article Title: Rare Helicobacter pylori Virulence Genotypes in Bhutan

    doi: 10.1038/srep22584

    Figure Lengend Snippet: Variation in the CagA amino acid sequence of East Asian-type and Western-type CagA. The target α-EAS sequences “AINRKIDRINKIASAGKG” in EPIYA segment D, based on the sequences of Japanese strains, are shown in frame. A sequence analysis revealed that the typical target sequences in Bhutanese strains was “(T/K)IN(G/R)KID(Q/R)(L/I)N(R/K)(T/I)ASA(G/N)KG,” where (X/Y) means X and Y as the two major amino acids. The sequences of the EPIYA-D segments in Bhutanese strains were highly variable compared to strains deposited in GenBank. In contrast, the East Asian-type and Western-type sequences of EPIYA-C segments in Bhutanese strains were largely identical, similar to the typical sequences of EPIYA-C segments deposited in GenBank. Reference strains used in Fig. 1A (strain name [accession number]) were 103a (AB110966.1), 105a (AB110967.1), 106a (AB110968.1), 108a (AB110969.1), 113b (AB110970.1), 120a (AB110971.1), 122b (AB110972.1), 125b (AB110973.1), 128a (AB110974.1), FJT77 (KF028580.1), 04-518 (AB267252.1), 03-166 (AB267253.1), 04-264 (AB267254.1), THP1477 (AB116744.1), 04-334 (AB267249.1), 03-292 (AB267250.1), 04-366 (AB267251.1), THP1260 (AB116742.1), M3 (AB116740.1), THP463 (AB116735.1), Korea23 (AB057044.1), Korea 12 (AB057043.1), K69 (FJ458129.1), Korea2-3 (AB057040.1), k266 (FJ458163.1), K265 (FJ458162.1), K264 (FJ458161.1), K261 (FJ458158.1), K260 (FJ458157.1), K259 (FJ458156.1). Reference strains used in Fig. 1C (strain name [accession number]) were India41 (AF222807.1), India99 (AF222809.1), OSC40A (EU089774.1), OSC42B (EU089775.1), PCR-156i (EU368669.1), PCR218vi (EU089766.1). RIGLD-OC149 (JX428784.1), SAN53 (EU089771.1), PD682 (EF450167.1), PD636 (EF450165.1), PD308 (EF450162.1), PD488 (EF450161.1), PD537 (EF450160.1), PD501 (EF450159.1), PD351 (EF450158.1), PD348 (EF450157.1), PD6481K (EF450153.1), 216G (GQ899171.1), 1407 (GU143415.1), HPI-14 (FJ849792.1), HPI-13 (FJ849791.1), HPI-11 (FJ849789.1), USA2791 (AB057099.1), Kazak3 (AB057098.1), USA35 (AB057095.1), Italy329 (AB057094.1), Arizona2 (AB057075.1), Arizona1 (AB057074.1).

    Article Snippet: Briefly, after antigen retrieval and inactivation of endogenous peroxidase activity, tissue sections were incubated with α-H. pylori antibody (DAKO, Glostrup, Denmark), anti-CagA antibody (b-300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or α-EAS Ab diluted 1:2,000 with diluting solution (DAKO) overnight at 4 °C.

    Techniques: Sequencing, Western Blot, Polymerase Chain Reaction

    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)

    Journal: Journal of Clinical Microbiology

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

    doi: 10.1128/JCM.02330-08

    Figure Lengend 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)

    Article Snippet: Alternatively, membranes were probed with a 1:5,000 dilution of rabbit IgG anti-CagA polyclonal antibody b-300 (Santa Cruz Biotechnology), followed by a 1:20,000 dilution of HRP-conjugated bovine anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology).

    Techniques: Expressing, Western Blot

    Analysis of the CagA protein patterns by immunoblot analysis. (A) Lysates of H . pylori isolated from the tissue specimens of various gastric patients were prepared as described in the Materials and Methods. Aliquots of protein (20 μg) were separated by SDS-gel electrophoresis and CagA was detected by immunoblotting using polyclonal anti-CagA antibody b-300 (Santa Cruz). The H . pylori 60190 and 26695 cagA knockout mutant (26695ΔA) were included as a positive and negative control, respectively, for CagA expression. The numbers at the top identify the various H . pylori colonies, while those on the left indicate the molecular weight markers in kDa. It can be seen that there are four types of CagA fragmentation patterns. (B) The percentages of ABD subtypes based on the CagA protein fragmentation patterns across the 96 H . pylori cagA ABD colonies that were analyzed and clearly ABD-2 is the predominant type.

    Journal: PLoS ONE

    Article Title: Fragmentation of CagA Reduces Hummingbird Phenotype Induction by Helicobactor pylori

    doi: 10.1371/journal.pone.0150061

    Figure Lengend Snippet: Analysis of the CagA protein patterns by immunoblot analysis. (A) Lysates of H . pylori isolated from the tissue specimens of various gastric patients were prepared as described in the Materials and Methods. Aliquots of protein (20 μg) were separated by SDS-gel electrophoresis and CagA was detected by immunoblotting using polyclonal anti-CagA antibody b-300 (Santa Cruz). The H . pylori 60190 and 26695 cagA knockout mutant (26695ΔA) were included as a positive and negative control, respectively, for CagA expression. The numbers at the top identify the various H . pylori colonies, while those on the left indicate the molecular weight markers in kDa. It can be seen that there are four types of CagA fragmentation patterns. (B) The percentages of ABD subtypes based on the CagA protein fragmentation patterns across the 96 H . pylori cagA ABD colonies that were analyzed and clearly ABD-2 is the predominant type.

    Article Snippet: Proteins (20 μg) were separated by SDS-gel electrophoresis, amounts of CagA and phosphorylated-CagA were determined by immunoblotting using polyclonal anti-CagA antibody b-300 (Santa Cruz) and anti-phosphotyrosine antibody PY99 (Santa Cruz).

    Techniques: Isolation, SDS-Gel, Electrophoresis, Knock-Out, Mutagenesis, Negative Control, Expressing, Molecular Weight