Cells extracts were performed in radioimmunoprecipitation assay (RIPA) buffer [100 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris pH 8] supplemented with protease (Roche) and phosphatase inhibitor cocktails (Sigma) and clarified by centrifugation. Proteins were analyzed by Western blot using the monoclonal anti-TWIST1 2C1a (Abcam), anti-p21CIP1 clone SX118 (Dako), and anti-HEB A9 (Santa Cruz Biotechnology) and the polyclonal anti-TWIST2 (proteintech), anti-E2A V18, anti-ITF2 K12, anti-H-RAS C20, anti-p15INK4B C20, anti-p16INK4A H156, p-histone H3 (Ser10)-R (Santa Cruz Biotechnology), and anti-GAPDH Abs16 (Millipore) antibodies and horseradish peroxidase–conjugated secondary antibodies (Dako). Antigen-antibody complexes were revealed with the Western-blotting Luminol reagent (Santa Cruz Biotechnology).
Analysis of TWIST1 by immunohistochemistry in mouse breast carcinomas was performed as described previously [7] (link) using the monoclonal anti-TWIST1 2C1a (Abcam).
Analysis of TWIST1 by immunohistochemistry in mouse breast carcinomas was performed as described previously [7] (link) using the monoclonal anti-TWIST1 2C1a (Abcam).