2.3. Western Blots

To measure the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), VEGF-C, VEGFR3, and LYVE-1, equal amounts of the samples were loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Then, the resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and incubated for one hour, at room temperature, in blocking solution (5% nonfat dried milk dissolved in Tris-buffered saline with Tween 20 [TBST] buffer). Then, the filters were probed, overnight, at 4°C, in blocking solution containing primary antibodies, diluted 1 : 1,000, against the following: IL-6 (MB9296, Bioworld), IL-1β (BS6067, Bioworld), TNF-α (11948S, Cell Signaling Technology), VEGF-C (sc-374628, Santa Cruz Biotechnology), VEGFR3 (ab27278, Abcam), LYVE-1 (ab14917, Abcam), and β-actin (AP0060, Bioworld). Membranes were washed twice with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology), for 1 hour, at room temperature, followed by washing three times. Signal detection was performed using an enhanced chemiluminescence substrate (Millipore, USA) and quantitated using Image J software.