Anti twist1

The following antibodies were used: rabbit polyclonal anti-Slug (1:1000), anti-Twist1 (1:500), anti-ZEB1 and anti-ZEB2 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). Rabbit monoclonal anti-E-cadherin (1:1000, Santa Cruz). Rabbit polyclonal anti-MDM2 (C-18) (1:500, Santa Cruz). Mouse monoclonal anti-α-tubulin (1:2000) and anti-fibronectin (1:1000) (Cell Signaling, Danvers, MA). Mouse monoclonal anti-N-cadherin (1:500), anti-ZO-1 (1:1000) and anti-vimentin (1:1000) (Abcam, Cambridge, UK). Mouse monoclonal anti-Restin (1:500), anti-His (1:1500) and anti-Flag (1:2000) (Sigma). Mouse monoclonal anti-p73 (1:1000), anti-p53 (1:1000) and anti-p63 (1:1000) (Abcam). Horseradish peroxidase–conjugated secondary antibodies were obtained from Amersham Biosciences.

Immunohistochemical staining of MTDH was performed following standard protocols. Briefly, paraffin-embedded tissue blocks were cut into 4-µm thick sections. The tissue sections were incubated with anti-MTDH antibodies (Cat#40-6500, Invitrogen, Carlsbad, CA, USA; dilution 1:400) overnight at 4°C, incubated in Envision^TM+Kits (Dako, Glostrup, Denmark), and visualized using 0.01% 3, 3-diaminobenzidine. The staining patterns were scored as follows: low expression: 0–50% of tumor cells with positive staining; high expression: more than 50% of tumor cells with positive staining. The intensity of staining of islet cells was used as an internal positive control. The score of immunohistochemical staining was evaluated independently by three investigators. Immunofluorescence staining was carried out as described previously [26 (link)]. The following primary antibodies are used in this study: anti-MTDH (Invitrogen; 1:50), anti-E-cadherin (Cat#610182, BD Biosciences, Franklin Lakes, NJ, USA; 1:50), anti-Twist1 (Cat#sc-81417, Santa Cruz, Dallas, TX, USA; 1:50), anti-GFP (Cat#ab13970, Abcam, Cambridge, UK; 1:250).