Anti snail sc 28199

Immunocytochemistry was done on PFA 4% fixed cells, and stained with the following primary antibodies: anti-E-Cadherin (BD Biosciences 610181), anti-SNAIL (sc-28199, Santa Cruz Biotechnology, INC.) and anti-ZO-1 (ZO-1 (C-19): sc-8146, Santa Cruz Biotechnology, INC.); the secondary antibodies were Alexa-Fluor 488 and Alexa-Fluor 594, from Molecular Probes. The nuclei were stained with NucRed^® Live 647 (Catalog number: R37106, Life Technologies), and preparations were analysed by confocal microscopy (Nikon A1).

Cells were washed twice on ice with PBS before adding protein lysis buffer (1× protease inhibitor cocktail, Roche, Basel, Switzerland, 1.5mM EDTA, 1mM DTT, 10% glycerol, 25mM HEPES, pH 7.6). The protein concentration was determined by the Bradford assay (BioRad, Hercules, CA) using BSA as a standard. Total protein (20μg) was resolved by 10% SDS-PAGE for subsequent western blot analysis using antibodies against the following proteins (diluted in TTBS (Tween–Tris Buffered Saline: 0.02% Tween-20 in 100mM Tris-CL [pH 7.5], 1:1000) as indicated): anti-p53 (DO7, DAKO, Glostrup, Denmark), anti-NKX2-1 (sc-53136, Santa Cruz, Santa Cruz, CA), anti-Sp1 (sc-14027, Santa Cruz, CA), anti-p65 (sc-8008, Santa Cruz, CA), anti-Snail (sc-28199, Santa Cruz, CA), anti –NF-Y (sc-17753, Santa Cruz, CA), anti-vimentin (sc-6260, Santa Cruz, CA), anti-IKKβ (sc-271782, Santa Cruz, CA), ant-α-tubulin (sc-5266, Santa Cruz, CA), anti-p21 (sc- 6246, Santa Cruz, CA) and anti-E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ). The gel was transferred to a Hybond-C Extra membrane (GE Healthcare, Little Chalfont, UK) and immunoblotted with primary antibody, as indicated in the figure legends. Anti-mouse or rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an ECL western blot detection system.