Journal: Journal of Biological Chemistry

Article Title: Elastase-released Epidermal Growth Factor Recruits Epidermal Growth Factor Receptor and Extracellular Signal-regulated Kinases to Down-regulate Tropoelastin mRNA in Lung Fibroblasts

doi: 10.1074/jbc.m200243200

Figure Lengend Snippet: FIG. 5. NE-initiated ERK activation is not mediated by release of bFGF, PDGF, or TGF-. Serum-starved lung fibroblasts (in 35-mm plates) were incubated for 30 min with 100 g/ml control, anti-bFGF, anti-PDGF, or anti-TGF--neutralizing antibodies and then challenged for 15 min with 10 g/ml NE or 10 ng/ml bFGF, PDGF, TGF-, EGF, or hbEGF as indicated. Status of ERK activation and total ERK amounts were determined by Western blot with anti-phospho-ERK and anti- ERK antibodies, respectively.

Article Snippet: Goat polyclonal anti-human TGF- (neutralizing, catalog number AF-239-NA) and goat polyclonal anti-human hbEGF (neutralizing, catalog number AF-259-NA) antibodies, recombinant human EGF, hbEGF, TGF- , betacellulin, amphiregulin (long form), and heregulin 1 (EGF domain) were obtained from R & D Systems.

Techniques: Activation Assay, Incubation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Elastase-released Epidermal Growth Factor Recruits Epidermal Growth Factor Receptor and Extracellular Signal-regulated Kinases to Down-regulate Tropoelastin mRNA in Lung Fibroblasts

doi: 10.1074/jbc.m200243200

Figure Lengend Snippet: FIG. 9. NE initiates ERK activation by releasing EGF. A, serum-starved lung fibroblasts (in 100-mm plates) were incubated with or without 5 g/ml NE or 5 g/ml PE for the indicated times, and then conditioned medium samples, as well as total cell lysates, were harvested in the presence of 1 mM DFP. Conditioned media samples (initial volume 12 ml) were concentrated (final volume 500 l) and dialyzed against 10 mM Tris-HCl, pH 7.5, at 4 °C on microconcentrators (Amicon), and 50 l of each sample were analyzed by Western blot (in non-reducing conditions) with anti-EGF neutralizing antibodies (upper part). The electrophoretic mobility of protein standards (Bio-Rad) is shown on the right. ERK activation status and total ERK in total cell lysates were determined by Western blot (in non-reducing conditions) with anti-phospho-ERK and anti-ERK antibodies, respectively (bottom part). B, serum-starved lung fibroblasts (in 12-well cluster plates) were incubated for 30 min in the presence of 100 g/ml control or anti-EGF-neutralizing antibodies (left part) or with or without 10 M AG1478 (right part). Then cells were challenged for 15 min with 100 l of the concentrated and dialyzed conditioned medium samples isolated from the control or NE-treated cells (A), 5 g/ml NE, 10 ng/ml EGF, 10 ng/ml TGF-, 10 ng/ml hbEGF, or 10 ng/ml betacellulin as indicated. Status of ERK activation and total ERK were determined by Western blot with anti-phospho-ERK and anti-ERK antibodies, respectively.

Article Snippet: Goat polyclonal anti-human TGF- (neutralizing, catalog number AF-239-NA) and goat polyclonal anti-human hbEGF (neutralizing, catalog number AF-259-NA) antibodies, recombinant human EGF, hbEGF, TGF- , betacellulin, amphiregulin (long form), and heregulin 1 (EGF domain) were obtained from R & D Systems.

Techniques: Activation Assay, Incubation, Western Blot, Control, Isolation