Alexa fluor 800 goat anti mouse or anti rabbit igg

The proteins were extracted from the transfected cells. Briefly, the cells were washed with PBS and suspended in RIPA lysis buffer (Thermo Scientific). The lysates were collected and stored at –20°C. Supernatant protein concentration was determined using the Bio–Rad protein assay system (Bio–Rad). Equal amounts of protein samples (100 μg) were fractionated by SDS/PAGE (8–15% polyacrylamide gels) and transferred to a nitrocellulose membrane. The blots were blocked for 2 h with 5% non-fat milk at room temperature, then immunoblotted with primary antibodies including Bax (1:200 dilution, Cell Signaling Technology), Bcl-2 (1:200 dilution, Cell Signaling Technology), caspase-3 (1:200 dilution, Cell Signaling), VEGFR-2 (1:200 dilution, Abcam), MEK (1:500 dilution, Cell Signaling), p-MEK (1:500 dilution, Cell Signaling), ERK (1:500 dilution, Cell Signaling), p-ERK1/2 (p44/p42) (1:500 dilution, Cell Signaling), p38 (1:200 dilution, Santa Cruz Biotechnology), p-p38 (1:500 dilution, Cell Signaling) in PBS and incubated at 4°C overnight. The membranes were washed with PBS-T and then incubated with secondary antibodies: Alexa Fluor 800 goat anti-mouse or anti-rabbit IgG (Invitrogen) and detected by the Odyssey v1.2 software by measuring the band intensity (area × OD) for each group. GAPDH was used as a loading control.

The total amount of protein was extracted from HUVECs, 4T1 breast cancer cells and tumor tissues in each group for immunoblotting analysis. Briefly, the protein concentrations were determined with a bicinchoninic acid protein assay kit using bovine serum albumin as the standard. Equal amounts of protein (100 μg) were fractionated by SDS-PAGE and blotted to PVDF membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% non-fat milk and incubated with the primary antibody at 4 °C overnight. Then the blots were incubated with secondary antibody: Alexa Fluor® 800 goat anti-mouse or anti-rabbit IgG (Invitrogen) for 1 h at room temperature. The primary antibodies against VEGFR2, Akt, phosphorylated of Akt (p-Akt), Bcl-2, Bax, MEK, p-MEK, ERK, p-ERK, Raf and GAPDH were purchased from Abcam and Cell Signaling. Western blot bands were captured by using the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified with Odyssey v1.2 software (LI-COR Biosciences). GAPDH was used as the internal control. Western blotting experiments were repeated four times.