Ccr 7

Transfected A549 cells were washed twice in ice-cold PBS and lysed in RIPA lysis buffer with protease inhibitors (50 nmol/l HEPES, pH 7.5, 150 nmol/l NaCl, 1% glycerol, 1% Triton, protease inhibitor cocktail). The protein concentration was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 25 µg protein was loaded per sample for SDS-PAGE (12% acrylamide) and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked using 5% skimmed milk in TBS-Tween (TBST; 0.05% Tween-20) at room temperature for 1 h, and then incubated with antibodies against VEGF (1:800; cat. no. 34-4300; Sigma-Aldrich; Merck KGaA), CCR-7 (1:2,000; cat. no. ab32527; Abcam) or tubulin (1:2,000; cat. no. SC-12462; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, the membrane was washed 3 times with TBST (10 min each), followed by incubation with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody for 1 h at room temperature (1:1,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.). The signal was captured using a Super Signal West Pico chemiluminescent substrate (cat. no. 34080; Pierce; Thermo Fisher Scientific, Inc.) to visualize the bands, and Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used for grayscale analysis.