Western blotting to analyze pathway inhibition

Tumors were isolated from euthanized, VIA-treated mice and immediately flash frozen. Tissue extracts were prepared as previously described [27]. Equal amounts of proteins (50 μg) were resolved by 4–15% gradient SDS PAGE. After transfer, membranes were probed with specific antibodies recognizing target proteins: pTyr (Sigma), ErbB2, ErbB3, Akt, pAkt473, Erk 1/2, pErk1/2 (Cell Signaling, Beverly, MA, USA), survivin, actin (Sigma, St. Louis, MO, USA), 4EBP-1, p4EBP-1, s6, ps6 (Santa Cruz Biotech, Santa Cruz, CA, USA), and then with IRDye 800 conjugated anti-rabbit or mouse IgG or Alexa Fluor 680 anti-rabbit IgG, and were visualized using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) as previously described [27].