P erk1 2 p44 p42

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.