Hrp conjugated second antibodies

After incubation with different groups of treatment for 48 h, the A549 and PC9 cells were harvested and split in RIPA lysis buffer with proteinase inhibitors. The protein concentration of extracts from tested cells were determined by BCA qualitative method. We resolved the extracts in 10% SDS-PAGE (Beyotime, Nanjing, China) and subsequently transferred them to PVDF membranes (Millipore, Bedford, MA, United States). After maintaining in 5% skim milk for 1 h, the PVDF membranes were incubated for 12 h at 4°C in 5% skim milk containing various primary antibodies (VEGFR2, VEGF, PI3K, p-PI3K, Akt, p-Akt 1:1000, caspase-3, Cleaved Caspase-3, Bax, Bcl-2 1:500, and GAPDH 1:5000). All the primary antibodies and horseradish peroxidase (HRP)-conjugated second antibodies were purchased from Cell Signaling Technology. Afterward, the PVDF membranes were washed with tris buffered saline containing 0.1% Tween-20 (TBST) for 10 min, three times, and incubated in HRP-conjugated second antibodies for 1 h. The membranes were washed again with TBST and visualized with ECL regents (Millipore, Bedford, MA, United States). Image J software (NIH image) was used to measure the densitometry of the protein bands.