Vimentin 2707 1

Fresh-frozen lymphnode specimens were broken into smaller pieces and then lysed in lysis buffer (1% SDS, 10 Mm Tris-HCl, pH 7.6, 100 mM phenylmethanesulfonyl fluoride) on ice for 15 minutes. Protein denaturation was performed at 95°C for 10 minutes, and then, lysates were centrifuged at 12,000 rpm at 4°C for 10 minutes followed by collection of the upper clear cell lysates. The protein concentration was measured by the Bradford method. Equal amounts of protein (60–90 µg/line) were loaded, separated by SDS-PAGE gels and blotted onto poly vinylidene difluoride (PVDF) membranes. GAPDH was used as an internal control. Antibodies against the following proteins were used: GAPDH (0411: sc-47724), OVOL2 (sc85803) (Santa Cruz Biotechnology, CA, USA), vimentin (2707-1) (Epitomics, Burlingame, CA, USA), E-cadherin (610181), Smad2/3 (610842) (BD Biosciences, San Jose, CA, USA), Snail (3879), p-AKT^Ser473 (4060), AKT (9272), p-Smad2/3 (8828), Smad2/3 (8685), p-ERK1/2 (Thr202/Tyr204) (4370), ERK1/2 (9102) (Cell Signaling Technologies, Danvers, MA, USA), POSTN (ab14041) (Abcam, Hong Kong, China). Goat anti-rabbit (Santa Cruz, sc-2004), goat anti-mouse (Santa Cruz, sc-2005) and donkey anti-goat (Santa Cruz, sc-2020) secondary antibodies were incubated with the membranes.