Skip to main content
Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Identification of Desmoglein-2 as a novel target of Helicobacter pylori HtrA in epithelial cells

Fig. 3

MMP and ADAM10 inhibitors do not block HpHtrA activity. A NCI-N87 cells were infected with H. pylori strains P12 wt, N6 wt and N6 ΔhtrA for 24 h. Where indicated, cells were treated with the broad range MMP inhibitor Batimastat (Bat) and ADAM10 inhibitor GI254023X (GI). Supernatants of infected cells were analyzed by gelatin zymography (upper panel) for proteolytic activity of activated MMPs and by casein zymography (lower panel) for proteolytic activity of secreted HpHtrA from H. pylori P12 wt and N6 wt. As a control, 10 µg of bacterial lysates of H. pylori P12 wt, N6 wt and N6 ΔhtrA were loaded on the zymograms. B NCI-N87 cells were stimulated for 24 h with 50 ng/ml TNFα to induce MMP activation. Where indicated, cells were additionally treated with broad range MMP inhibitor Batimastat (Bat), ADAM10/17 inhibitor GI254023X (GI) or a combination of both (Bat/GI). As a control, cells were incubated with 0.1% of the solvent DMSO. Supernatants of NCI-N87 cells were analyzed by Western blot for N-terminal fragments of hDsg2 (Dsg2NTF) and hCdh1 (Cdh1NTF) using antibodies recognizing the extracellular domains of hDsg2 and hCdh1. The overlay of hDsg2 (green) and hCdh1 (red) is presented to exclude possible antibody cross reactions. C 50 ng rCdh1 were incubated with 100 ng recombinant human MMP-7 for 16 h at 37 °C. As indicated, increasing concentrations of the broad range MMP inhibitor Batimastat (Bat) were added. Proteins were separated by SDS-PAGE and analyzed by Western blot. Full length rCdh1 (rCdh1FL) and cleavage fragments were detected using an antibody recognizing the extracellular domain of hCdh1. D 50 ng rCdh1 was incubated with 250 ng HpHtrA wt, inactive mutant (SA) or 100 ng active MMP-7 for 16 h at 37 °C. Where indicated broad range MMP inhibitor Batimastat (Bat), ADAM10 inhibitor GI254023X (GI) or a combination of both (Bat/GI) was added. Proteins were separated by SDS-PAGE and analyzed by Western blot. Full length rCdh1 (rCdh1FL) and cleavage fragments were detected using an antibody recognizing the extracellular domain of hCdh1. HpHtrA was detected with a polyclonal HpHtrA antibody

Back to article page