C myc

Cells were lysed in ice-cold RIPA buffer (50 mM of Tris-HCl pH 7.4, 150 mM of NaCl, 1% Triton X-100, 0.25% deoxycholate, 1.5 mM of MgCl₂, 1 mM of EGTA, 1 mM of phenylmethylsulfonyl fluoride, 10 mM of ZnAF, 10 mM of pervanadate, 10 µg/ml of leupeptin and 10 µg/ml of aprotinin) and incubated for 30 min. The protein concentration was determined using the BCA method. The cell lysate was heated at 100°C for 10 min after being mixed with sample loading buffer. The protein samples were equally loaded on 10% SDS-PAGE, and transferred onto nitrocellulose membranes. After being blocked with 5% non-fat milk in Tris-buffer, the membrane strips were incubated with a primary antibody overnight at 4°C, followed by Alex Fluor 680/790-labeled goat anti-rabbit or goat anti-mouse IgG (Li-COR Biosciences, Lincoln, NE, USA). The strips were visualized using the LI-COR Odyssey Infrared Imaging System (Li-COR Biosciences).
The primary antibodies were as follows: p38, phospho-p38, ERK, phospho-ERK, JNK and phospho-JNK (1:1,000; Cell Signaling Technology, Danvers, MA, USA); MMP2, VEGF, EGFR, cyclin D and E (1:1,000; Abcam, Cambridge, MA, USA); p65, Bcl2, Snail, c-MYC, SSRP1 and GAPDH (1:1,000; GeneTex, Inc.).