Rabbit polyclonal anti n cadherin

Tumor cells (A549, or A549/DDP) were seeded into six-well plates (2.5 ×
10⁵/well). After 24 h culture, the cells were treated with bFGFmAb 100µg/mL or
PBS by adding them directly into the culture medium. On the next day, the cells were lysed
with RIPA lysis buffer for 30 min at 4°C and centrifuged at 12,000 g for 15 min. A pierce
BCA Protein Assay Kit (Thermo Fisher Scientific) was used to determine protein
concentrations following the manufacturer’s instructions. The protein was separated by 10%
SDSPAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA,
USA). After blocking with 5% blotto (skimmed milk) in Tris-buffered saline and Tween 20
for 1 h, the primary antibodies rabbit monoclonal anti-bFGF (CST, USA; 1:1000 dilution),
mouse monoclonal anti-E-cadherin (CST, 1:1000 dilution), rabbit polyclonal anti-N-cadherin
(Abcam, UK; 1:1000 dilution), rabbit polyclonal anti-LRP (Santa Cruz, USA, 1:200
dilution), and mouse monoclonal anti-β-actin (CST, USA, 1:1000 dilution) were added and
incubated at 4°C overnight. After incubation with the secondary antibody goat anti-rabbit
IgG-HRP (GE Healthcare, 1:1000 dilution), the bound antibodies were detected using the ECL
Plus Western Blotting Detection system (Life Technologies, USA). Finally, the intensities
of the bands were analyzed using the Bandscan Software.