Ecl plus western blot system kit

In order to investigate expression level of ERK3, TNF-α, VEGF-C and VEGFR-3, hLEC cells were collected and lysed by RIPA lysis buffer (Cell Signal Technology, Danvers, MA, USA) on ice according to the manufacturer’s instruction. Then, the total cellular proteins were subjected to SDS-PAGE (10%) for western analysis. After transferring to polyvinylidene difluoride (PVDF) membranes, blots were incubated with 5% BSA (Gibco, Rockville, MD, USA) in Tris buffered saline containing 0.5% Tween 20 for 60 min. Then incubated overnight at 4 °C on a rocker with the following primary antibodies: anti-ERK3 (SAB, #33914), anti-TNF-α (abcam, #ab6671), Anti-VEGF antibody (SAB, #41702) and VEGFR3 (SAB, #44895) antibody (1:1,000). Following washing three times with TBST for 5 min, membranes were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG polyclonal secondary antibody (1:3,000) (Beyotime, #A0216, Beijing, China) at room temperature for 1 h. Using Amersham’s ECL + plusTM Western blot system kit for color developing. Signals were detected with enhanced chemiluminescence, using GAPDH as the internal standard (Kodak, Rochester, NY, USA).