Journal: The FEBS journal

Article Title: Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells.

doi: 10.1111/j.1742-4658.2005.05035.x

Figure Lengend Snippet: Fig. 5. HAT induces biphasic activation of ERK through AR-depend- ent and -independent pathways. (A, C) NCI-H292 cells were pre- treated with the vehicle alone (Veh), PD98059 (PD; 10 lM), anti-AR neutralizing antibody (aAR; 10 lgÆmL)1) or normal mouse IgG1 (IgG; 10 lgÆmL)1) for 20 min. The cells were then stimulated with HAT (200 nM) for 5 min (A) or the indicated durations (C) in the presence of the indicated inhibitors or antibody. (B) For the 480 min culture period, NCI-H292 cells were stimulated with HAT for 30 min and then treated with the vehicle alone or with PD98059 (PD; 10 lM), anti-AR neutralizing antibody (aAR; 10 lgÆmL)1) or normal mouse IgG1 (IgG; 10 lgÆmL)1) and further cultured for 480 min. (D) Cells were stimulated with AR (3 lgÆmL)1) for the indicated durations. (E) Cells were stimulated with increas- ing concentrations of HAT for 5 min. Immunoblotting, with repea- ted probing using the antibodies indicated on the left side of the figure, was used to analyse the cell lysates.

Article Snippet: Human recombinant AR, anti-TACE mAb (clone 111633) and nonimmune mouse IgG1, used as negative controls, were from R&D Systems Inc. (Minneapolis, MN, USA).

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: The FEBS journal

Article Title: Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells.

doi: 10.1111/j.1742-4658.2005.05035.x

Figure Lengend Snippet: Fig. 8. TACE is involved in HAT-induced AR release, which pro- longs HAT-induced AR gene expression by positive feedback loop. (A, D, E) NCI-H292 cells were pretreated with the vehicle alone (Veh), GM6001 (GM; 3 lM), TAPI-1 (TAPI; 3 lM), PD98059 (PD; 10 lM) or anti-AR neutralizing antibody (aAR; 10 lgÆmL)1) for 20 min. (B, C) NCI-H292 cells were transfected with siRNA for TACE or control siRNA (cont) and cultured for 72 h. (A, C) Cells were then stimulated with HAT (200 nM) for 2 h and ELISA was used to determine the AR concentration in culture supernatant. (B) Cells were then collected and stained with anti-TACE antibody or normal mouse IgG1 (background) and analysed for cell surface TACE density by flow cytometry. (D, E) Cells were then stimulated with AR (3 ngÆmL) for 1 h (D) or HAT (200 nM) for 2 h (E) and the total RNA was extracted, and quantitative real-time RT ⁄ PCR (TaqManTM) analysis was used to determine the amounts of AR and b-actin mRNA. The results are presented as the mean ± SD (n ¼ 3). *P < 0.05, **P < 0.01 when compared with vehicle-treated cells and ##P < 0.01 when compared with HAT (A, C) or AR (B)-treated cells in the absence of inhibitors, Dunnett’s test.

Article Snippet: Human recombinant AR, anti-TACE mAb (clone 111633) and nonimmune mouse IgG1, used as negative controls, were from R&D Systems Inc. (Minneapolis, MN, USA).

Techniques: Gene Expression, Transfection, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Cytometry