Horseradish peroxidase hrp conjugated goat anti rabbit igg polyclonal secondary antibody

Total protein lysates from the melanoma cells were prepared by incubation with the RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) on ice according to the manufacturer’s instructions. Then, equal amounts of total protein lysates were separated on a 10% SDS-PAGE. The separated proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% BSA (Gibco, Grand Island, NY, USA) in Tris-buffered saline (TBS) containing 0.5% Tween-20 (TBST) for 60 min. The blots were then incubated overnight at 4° C with primary antibodies against VEGFA (1:1000, Abcam, CA, USA), cleaved caspase-3 (1:1000, Abcam), α-SMA (Abcam, 1:1000), E-cadherin (Abcam, 1:1000), vimentin (Abcam, 1:1000), and β-actin (Abcam, 1:1000). Then, after washing three times with 1X TBST buffer for 5 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG polyclonal secondary antibody (1:5000; Beyotime Biotechnology, Shanghai, China) at room temperature for 1 h. The blots were developed with the ECL+Plus chemoluminescence western blot system kit (Amersham, Cytiva, Shanghai, China). The density of the protein bands was measured using the ImageJ software.