Anti vimentin monoclonal antibody

Cells (~2×10⁴/sample) were seeded on glass coverslips and cultured for 24 hrs in growth medium. Then, slides were washed with PBS, fixed with 2.5% formaldehyde in PBS for 10 min at 4°C and incubated overnight at 4°C with 2 μg/mL R4 anti-uPAR monoclonal antibody or rabbit anti-uPAR_84-95 polyclonal antibody [30 (link)]. HPMCs plated on glass slides (30%-40% confluence), were fixed and permeabilized with 2.5% formaldeyde-0.2% Triton X-100 in PBS for 10 min at 4°C, then incubated with 2 μg/mL anti-vimentin monoclonal antibody (Dako), anti-cytokeratin 8/18 polyclonal antibody (MyBiosurce), or anti-von Willebrand factor VIII monoclonal antibody (Dako) for 1 hr at 4°C. Then, 1:800 goat Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 488-conjugated F(ab')2 fragment of rabbit anti-mouse IgG, or Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes) were applied to slides at 23°C for 40 minutes. After nuclear staining with 4-6-diamidino-2-phenylindole dye (DAPI), coverslips were mounted using 20% (w/v) mowiol and analyzed by a fluorescence inverted microscope connected to a video-camera (Carl Zeiss). To analyze FPR internalization, cells grown on glass slides were exposed to 10 nM N-formyl-Nle-or Leu-Phe-Nle-Tyr-Lys-fluorescein (Molecular Probes), diluted in serum-free DMEM for 30 minutes at 37°C as described [32 (link)].

Sample protein concentrations were measured by the Bradford protein assay (Bio-Rad), and 20 μg total protein/lane was subjected to electrophoresis on 10% polyacrylamide gels followed by electroblotting onto nitrocellulose filters. The membranes were blocked with 3% milk in TBS-T and incubated overnight at 4°C with the following antibodies: anti-NUAK1 polyclonal antibody (Cell Signaling Technology, #4458), anti-E-cadherin monoclonal antibody (BD Transduction Laboratories, 610181), anti-N-cadherin monoclonal antibody (BD Transduction Laboratories, 610920), anti-SNAI1 polyclonal antibody (Cell Signaling Technology, #3879), anti-SNAI2 polyclonal antibody (Cell Signaling Technology, #9585), anti-ZEB1 antibody (Santa Cruz Biotechnology, sc-25388), anti-vimentin monoclonal antibody (Dako, #M0725), and anti-β-actin polyclonal antibody (Sigma-Aldrich, A5441). The membranes were then washed with TBS-T and incubated with specific secondary antibodies, and the proteins were visualized using the ECL western blotting detection system (GE Healthcare).