E cadherin h 108

17β-estradiol (E2), 4-hydroxytamoxifen (OHT), and tunicamycin were purchased from Sigma-Aldrich. E2 and OHT were dissolved in ethanol, tunicamycin in dimethyl sulfoxide (DMSO). Primary antibodies were the following: against the FLAG tag (M2, Sigma Aldrich), α-tubulin (DM1A, Sigma Aldrich), histone H3 (ab1791, Abcam), pERK1/2 (E-4, Santa Cruz Biotech), ERK2 (C-14, Santa Cruz Biotech), GAPDH (ab9484, Abcam), ERα (HC-20, Santa Cruz Biotech), GPER (LS-A4272, LifeSpan Biosciences), vimentin (V9, Abcam), and E-cadherin (H-108, Santa Cruz Biotech).

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors for 20 min. Proteins (20 μg) extracted from cells were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with blocking buffer (5% non-fat milk in PBS containing 0.1% Tween 20) and then incubated with primary antibodies dissolved in blocking buffer at 4 ℃ overnight. Membranes were washed with TBS-T buffer (PBS with 0.1% Tween 20) for 5 min three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The membrane was developed using an ECL Kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) with X-ray film.
The primary antibodies used in this western blotting were as follows: E-cadherin (H-108, 1:1000, sc-7870; Santa Cruz Biotechnology, Inc.), N-cadherin (H-63, 1:1000, sc-7939; Santa Cruz Biotechnology, Inc.), fibronectin (EP5, 1:1000, sc-8422; Santa Cruz Biotechnology, Inc.), vimentin (V9, 1:1000, sc-6260; Santa Cruz Biotechnology, Inc.), slug (1:1000, GTX31749; GeneTex), snail (1:10000, GTX125918; GeneTex), and GAPDH (D4C6R, 1:1000, 97,166; Cell Signaling Technology).