Quantitative Analysis of Protein Expression

Western blot was performed according to a previously described method^{18 (link)}. GAPDH was used as an internal loading control. Densitometric analysis was performed on western blot images using Image Pro-Plus 6.0 software (Media Cybernetics). The catalog numbers and dilution of the antibodies used (Abcam, Cambridge, MA, USA) are as follows: matrix metalloproteinase (MMP)-2, ab37150, 1:500; MMP-9, ab38898, 1:1000; E-cadherin, ab15148, 1:500; vimentin, ab92547, 1:2500; PDK1, 1:800, ab110025; p-AKT1 (phospho S473), 1:2500, ab194201; p-mTOR (phospho S2448), 1:1000, ab109268; AKT1, 1:1000, ab227100; mTOR, ab2732, 1:2000; and GAPDH, ab9485, 1:2500. Western blot assays were replicated three times.