Journal: FEMS microbiology letters

Article Title: YWHAE is a novel interaction partner of Helicobacter pylori CagA.

doi: 10.1093/femsle/fnx231

Figure Lengend Snippet: Figure 1. Screening of YWHAE interacted with CagA by the GAL4 yeast two- hybrid method. (a) Western blot detection of the fusion protein GAL4 BD-CagA in AH109 yeast. Proteins were subjected to SDS-PAGE for Western blot analy- sis. GAL4 DNA-BD monoclonal antibody was employed. (b) The yeast two-hybrid screening by yeast mating. In the screening test, 73 heterozygotes were obtained, growing well and showing green on SD/-Ade/-His/-Trp/-Leu/X-α-gal plates con- taining 3-AT. (c) β-gal assays (of the heterozygotes obtained) to qualitatively eval- uate the phenotype of the heterozygotes. Positive, e.g. ∗2, ∗6, ∗15, ∗24, ∗35, ∗48,

Article Snippet: The clear lysates were mixed with anti-CagA (b-300) antibody (5 μg, Product no. sc-25766) and Protein A&GAgarose (20 μL), and the resultant immunoprecipitation complexes were analyzed byWestern blot using anti-CagA (b-300) antibody (1:200 dilution) and antiYWHAE antibody [14-3-3ε (T-16), 1:200 dilution] (all antibodies and agarose were obtained from Santa Cruz Biotechnology).

Techniques: Western Blot, SDS Page, Two Hybrid Screening

Journal: FEMS microbiology letters

Article Title: YWHAE is a novel interaction partner of Helicobacter pylori CagA.

doi: 10.1093/femsle/fnx231

Figure Lengend Snippet: Figure 2. Interaction between CagA and YWHAE in vitro. (a) GST and GST-CagA fusion proteins expressed bacterially were purified via glutathione-Sepharose beads. (b) GST pull-down assay. 35S-labeled YWHAE translated in vitro was incu- bated with GST or GST-CagA. The binding proteins were separated by SDS-PAGE and probed by autoradiography.

Article Snippet: The clear lysates were mixed with anti-CagA (b-300) antibody (5 μg, Product no. sc-25766) and Protein A&GAgarose (20 μL), and the resultant immunoprecipitation complexes were analyzed byWestern blot using anti-CagA (b-300) antibody (1:200 dilution) and antiYWHAE antibody [14-3-3ε (T-16), 1:200 dilution] (all antibodies and agarose were obtained from Santa Cruz Biotechnology).

Techniques: In Vitro, Purification, Pull Down Assay, Labeling, Binding Assay, SDS Page, Autoradiography

Journal: FEMS microbiology letters

Article Title: YWHAE is a novel interaction partner of Helicobacter pylori CagA.

doi: 10.1093/femsle/fnx231

Figure Lengend Snippet: Figure 4. CagA enhances YWHAE-mediated NF-κB activation. (a) The SGC7901 or AGS cells were co-transfected with pCMVTNT-CagA and pCMVTNT-YWHAE. pNFκB-luc was employed as a reporter vector, and pRL-TK was employed to nor- malize each transfection efficiency. The transfection of the empty pCMVTNT vector served as a negative control. After transfection for 48 h, the cells were harvested. The relative luciferase activities were detected. Each transfection was performed in duplicate. (b) Detection of YWHAE expression with YWHAE siRNA. The SGC7901 or AGS cells were transfected with YWHAE siRNA. Subse- quently, the expression of YWHAE protein was probed by Western blot with an anti-YWHAE antibody. (c) The SGC7901 or AGS cells were co-transfected with pCMVTNT-CagA and YWHAE siRNA. pNFκB-luc and pRL-TK were employed with the same function as above. The transfection of the empty pCMVTNT vector and the NC-siRNA served as negative controls. After the transfection for 72 h, the cells were harvested. The relative luciferase activities were detected. Each trans- fection was performed in duplicate. All data were showed as the mean ± SD of five experiments. In addition, each P value was derived from a paired-samples t-test; meanwhile P < 0.05 was considered statistically significant. The single asterisk denotes P < 0.05 for comparisons with the ‘CMVTNT’ group, and the double asterisks denote P < 0.05 for comparisons with the corresponding group directed in each figure.

Article Snippet: The clear lysates were mixed with anti-CagA (b-300) antibody (5 μg, Product no. sc-25766) and Protein A&GAgarose (20 μL), and the resultant immunoprecipitation complexes were analyzed byWestern blot using anti-CagA (b-300) antibody (1:200 dilution) and antiYWHAE antibody [14-3-3ε (T-16), 1:200 dilution] (all antibodies and agarose were obtained from Santa Cruz Biotechnology).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Negative Control, Luciferase, Expressing, Western Blot, Derivative Assay

Journal: FEMS microbiology letters

Article Title: YWHAE is a novel interaction partner of Helicobacter pylori CagA.

doi: 10.1093/femsle/fnx231

Figure Lengend Snippet: Figure 3. Interaction between CagA and YWHAE in vivo. (a) The co-localization of CagA and endogenous YWHAE. SGC7901 and AGS cells infected with H. py- lori NCTC 11637 were stained with anti-CagA (green) and anti-YWHAE (red), and then observed by confocal fluorescence microscopy. (b) Co-IP detection of inter- action between CagA and endogenous YWHAE. SGC7901 cells were infected by H. pylori NCTC 11637 or 11637 cagA. Afterwards, the cell lysates were immuno- precipitated with anti-CagA followed by Western blot analysis with anti-CagA or anti-YWHAE. (c) and (d) Co-IP detection of interaction between CagA and over- expression YWHAE. The Cos-7 or AGS cells were co-transfected by the CagA and YWHAE expression vectors. (c) The Cos-7 cells’ lysates were then immunopre- cipitated with anti-Flag followed by Western blot analysis with anti-Flag or anti- Myc. (d) The AGS cells’ lysates were then immunoprecipitated with anti-CagA followed by Western blot analysis with anti-CagA or anti-YWHAE. wt, wild type; IP, immunoprecipitation; WB, Western blot.

Article Snippet: The clear lysates were mixed with anti-CagA (b-300) antibody (5 μg, Product no. sc-25766) and Protein A&GAgarose (20 μL), and the resultant immunoprecipitation complexes were analyzed byWestern blot using anti-CagA (b-300) antibody (1:200 dilution) and antiYWHAE antibody [14-3-3ε (T-16), 1:200 dilution] (all antibodies and agarose were obtained from Santa Cruz Biotechnology).

Techniques: In Vivo, Infection, Staining, Fluorescence, Microscopy, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Transfection, Expressing, Immunoprecipitation