Imaging analyzer

Tissue lysates were prepared by pipetting cell lysis buffer (cell signaling; 20 mM Tris-HCL/150 mM NaCl/1 mM Na₂EDTA/1 mM EGTA/1% Triton/2.5 mM sodium pyrophosphate/1 mM β-glycerophosphate/1 mM NaVO/1 µg/mL leupeptin) with 1 mM phenylmethylsulfonyl fluoride separated on 12.5% polyacrylamide gels (50 µg per lane), and transferred to immobilon polyvinylidene difluoride membrane. The blots were probed with PGEs-1 monoclonal antibody at 1:1,000 dilution in 5% bovine serum albumin, and then anti-β-actin antibody was prepared at 1:10,000 dilution in 5% milk, developed using peroxidase-conjugated goat anti-mouse antibodies, and visualized using an ECL plus detection kit (Amersham Pharmacia Biotech, Inc. Piscataway, NJ, USA), following the manufacturer's instructions and then exposed to Kodak CP-BU film (Rochester, NY, USA). Expression level of mPGEs-1 was normalized to β-actin and quantified using imaging analyzer (Bio-Rad Laboratories, Hercules, CA, USA).

Total proteins were extracted from both cell types using RIPA lysis buffer containing protease-phosphatase inhibitors (ab201119, abcam, the UK), and PMSF (Solarbio, Beijing, China) and placed on ice for 15 min. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked with 5% skim milk in tris-buffered saline containing 0.2% Tween-20 (TBST) for 40 min and were then treated with monoclonal rabbit antibodies anti TRAF5 (1:400, ProteinTech, Wuhan, China), IKβ-α (1:200, ProteinTech, Wuhan, China), N-cadherin (1:200, Beyotime, Nantong, China), Vimentin (1:200, Beyotime, Nantong, China) and MMP7 (1:200, Beyotime, Nantong, China), and monoclonal mouse GAPDH (1:1,000, ThermoFisher, Carlsbad, USA) and incubated overnight at 4 °C. After washing three times with TBST for 15 min, membranes were incubated with the secondary antibody for 40 min at room temperature. After washing three times with TBST for 10 min, the immunoreactive bands were visualized using ECL Plus kit (Beyotime, Nantong, China) and the imaging analyzer (Bio-rad, USA) was used for observation and image acquisition. GAPDH was used as a loading control.