SNAIL Polyclonal Antibody

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	-Rabbit (1:100)

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-Rabbit (1:100)

Publication protocol

After two days of treatment with 10−7 M MART-10 or 1α,25(OH)2D3, cells were washed once with PBS and lyzed in the lysis buffer containing 50 mM Tris-HCl, 50 mM β-glycerol phosphate, 50 mM NaCl, 1 mM Na3Vo4, 1 mM EDTA, 1 mM EGTA, 1% NP40, and freshly added 1 mM DTT, 1 mM PMSF, 2 μg/mL aprodenin, 2 μg/mL leupeptin, and 2 μg/mL pepstatin right before lysis. The antibodies used in this experiment were monoclonal antibodies against E-cadherin (1:1000, #3195, Cell Signaling Technology, Irvine, CA, USA), N-cadherin (1:1000, #13116, Cell Signaling Technology), P-cadherin (1:1000, #2189, Cell Signaling Technology), Zeb1 (1:500, TA802313, OriGene Technologies, Inc., Rockville, MD, USA), Zeb 2 (1:500, TA802113, OriGene Technologies), Snail (1:100, PA5-23472, Thermo Fisher Scientific, Waltham, MA, USA), Slug (1:1000, #9585, Cell Signaling Technology), Twist (1:100, sc-15393, Finnell Street Dallas, TX, USA), LCN2 (1:500, PAB9543, Abnova, Taipei, Taiwan). After washing in TBST, the blots were detected using ECL reagents (Millipore, WBKLS0500, Temecula, CA, USA). The second body was goat anti-mouse/rabbit IgG conjugated with HRP (Cell Signaling Technology, Boston, MA, USA). The incubation time for primary or secondary antibody was 2 or 1 h, respectively, under room temperature. 10% SDS-PAGE and Tris-Glycine buffer system were applied. PVDF membrane and wet transfer (400 mA, 2 h) were used. Membranes were detected and analyzed by VersaDoc™ Imaging System (Bio-Rad, Hercules, CA, USA). Expression of targeted protein relative to tubulin (as the loading control) was calculated. The detailed procedure was described previously [30].

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