Anti slug clone l40c6

Cells grown on glass coverslips were fixed and permeabilized in phosphate-buffered saline (PBS) containing 2% paraformaldehyde and 1% Triton X-100, while sections were dewaxed, hydrated through decreasing concentrations of ethanol, rinsed in distilled water, and subjected to antigen retrieval treatment. Samples were washed in PBS and then incubated in PBS and 1% bovine serum albumin (Sigma-Aldrich) to block nonspecific binding, before incubating with the primary anti-SLUG (clone L40C6, Cell Signaling Technology) and anti-E-cadherin (clone 32A8, Cell Signaling Technology) antibodies diluted in PBS and 1% bovine serum albumin overnight at 4 °C. Samples were subsequently rinsed in PBS and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Dako). Mounting and nuclei counterstaining were performed using the ‘pro long antifade reagent with DAPI' (Invitrogen, Carlsbad, CA, USA) and observed under a fluorescence microscope (Carl Zeiss, Milan, Italy).

The immunoblotting, isolation of nuclear protein fractions and immunoprecipitation procedures were performed as previously described.^{11 (link)} Antibodies used in this work were as follows: anti-p53 (clone BP53-12, Novocastra), anti-β-actin (clone AC-74, Sigma-Aldrich), anti-MDM2 (clone SMP14 and clone H-221, Santa Cruz Biotechnology), anti-L11 (clone 3A4A7, Invitrogen), anti-SLUG (clone L40C6, Cell Signaling Technology), anti-E-cadherin (clone 32A8, Cell Signaling Technology), anti-c-Myc (Cell Signaling Technology) and anti-Lamin B (C-20, Santa Cruz Biotechnology).