Polyclonal anti vimentin

Binding of ve-cadehrin to vimentin or P-vimentin was assayed in endothelial cells cultured in CLP or SHAM serum for 24 hrs or pretreated with 5-HT at indicated concentrations for 45 min at room temperature (RT). The cells were lysed in IP buffer (55 mM triethylamine [pH 7.5], 111 mM NaCl, 2.2 mM EDTA, and 0.44% SDS + 1% Triton X-100, 1 mM PMSF, PIM) and precleared by incubation with nonimmune rabbit serum and protein A for 1 hr and then centrifuged as described previously16 (link)20 (link)22 (link)24 (link). The precleared lysate was combined with an equal volume of a 1:1 slurry of protein A Sepharose beads and mixed overnight at 4⁰ C with a monoclonal ve-cadherin-Ab (Santa Cruz Dallas TX). Samples were separated on an SDS-PAGE and analyzed by WB with polyclonal anti-vimentin (Santa Cruz, Dallas TX) or P-vimentin (21st Century Biochemicals, Inc. Marlboro MA). The signals were developed with the ECL detection system.
For RIP, endothelial cells were treated with 20 μM cold and [³H]-5HT (100 μCi/ml) in PBS/CM at RT. After 1 hr, the cells were washed quickly with ice cold PBS and harvested, and Rac was IP using monoclonal Rac-Ab as described22 (link). Immune precipitates were eluted from the beads by incubation in SDS sample buffer, analyzed by 12.5% SDS-PAGE, and visualized by fluorography.

Catechol (1,2-dihydroxybenzene) was obtained from Sigma Aldrich (St. Louis, MO), prepared 100 mM stock solution in DMSO, and placed in dark at −20°C. Recombinant human epidermal growth factor (EGF) was purchased from Prospec (East Brunswick, NJ), dissolved at a concentration of 100 μg/mL in sterile water containing 0.1% bovine serum albumin (BSA), and placed in deep freezer at −70°C. Polyclonal anti-E-cadherin antibody was purchased from BD Biosciences (San Jose, CA). Polyclonal anti-Vimentin, anti-phospho-EGFR, anti-EGFR, anti-β-actin, anti-CD44, and anti-Nanog antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX). Monoclonal anti-Snail, anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK antibodies were obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX).