Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of EGFR signaling abrogates smooth muscle proliferation resulting from sustained distension of the urinary bladder.

doi: 10.1038/labinvest.3700483

Figure Lengend Snippet: Figure 4 ZD1839 inhibits stretch-induced SMC EGFR phosphorylation ex vivo. (a) Bladders were distended ex vivo for 12 h in the presence or absence of ZD1839 (10 mM) and EGFR phosphorylation was evaluated by IHC. The following conditions were tested: (a) No ZD1839, (b) Intraluminal ZD1839 (‘Inside’ in figure), (c) intraluminal þ extraluminal ZD1839 (inside þ outside), (d) extraluminal ZD1839 (Outside). Representative images from conditions (a) and (b), and are shown (magnification 200). (b) Image analysis of the intensity of p-EGFR staining under conditions (a–d). IHC staining intensity is scored in arbitrary units. The difference in p-EGFR levels between untreated bladders and those treated with ZD1839 was statistically significant (Po0.0001). There was no significant difference in the level of p-EGFR between bladders receiving ZD1839 by different routes (b–d). Significant values (Po0.05) are demarcated with an asterisk. (c) NRK-52E cells were serum-depleted for 24 h, preincubated for 30 min with ZD1839 (0.01–50.0 mM), then treated with recombinant EGF (100 ng/ml) for 10 min on ice, 5 min at 371C. Plasma membrane fractions were isolated, resolved by SDS-PAGE, and probed for total and phosphorylated EGFR. Untreated cells, and EGF-treated cells that did not receive ZD1839 were included as controls. The phospho-EGFR signal was almost completely abolished with 0.1 mM ZD1839. Ponceau S staining of the nitrocellulose membrane indicates equivalent protein loading.

Article Snippet: For all immunoblots neonatal rat kidney epithelial cells (NRK-52E) treated with or without 100ng/ml recombinant epidermal growth factor (rEGF, R&D Systems, Minneapolis, MN, USA) served as a positive control.

Techniques: Phospho-proteomics, Ex Vivo, Staining, Immunohistochemistry, Recombinant, Clinical Proteomics, Membrane, Isolation, SDS Page