Mouse anti e cadherin

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections; 5-μm thick sections were used. The biopsy paraffin-embedded tissue array was prepared. MT1-MMP was detected using rabbit monoclonal primary antibody (EPITOMICS) at a dilution of 1:400. Slides were incubated with the following primary antibodies overnight at 4 °C: mouse anti-E-cadherin (1:100; DAKO System), anti-vimentin (1:800; DAKO System), anti-N-cadherin (1:400; Abcam), anti-snail (1:800; Abcam), and anti-slug (1:400; Abcam). The specificity of the immunostaining reaction was verified by referring to previously confirmed positive- and negative-control tissue sections. Immunohistochemical staining was assessed by scoring, as described previously18 (link). All patients in this study provided informed consents, and the study was approved by the institutional ethics committee at the First Affiliated Hospital of Shihezi University School of Medicine.

Cells were lysed in RIPA buffer (Merck Millipore, Vimodrone, MI, Italy) containing PMSF (Sigma-Aldrich, Saint Louis, Missouri, USA), sodium orthovanadate (Sigma-Aldrich), and protease inhibitor cocktail (Calbiochem, San Diego, CA, USA), sonicated and centrifuged 15 min at 14,000 rpm at 4 °C. Equal amounts of protein were separated on Bolt^® Bis-Tris Plus gels, 4–12% precast polyacrylamide gels (Life Technologies, Monza, Italy). Fractionated proteins were transferred to a PVDF membrane using the iBlot 2 System (Life Technologies). Following 1 h blocking with Odyssey blocking buffer (Dasit Science, Cornaredo, MI, Italy), membrane was probed overnight at 4 °C with mouse anti -N-Cadherin and mouse anti-E-Cadherin (1:1000, DAKO, Glostrup, Denmark); rabbit anti-EGFR, (1:500 Cell Signalling Technology, Danvers, MA, USA); rabbit anti-PDGFR, rabbit anti p-p70S6k, rabbit anti-pERK, rabbit anti-ERK (1:1000, Cell Signalling Technology, Danvers, MA, USA), and 1 h at room temperature with goat anti-rabbit IgG Alexa Flour 750 antibody (Invitrogen, Monza, Italy). Membrane was visualised by the Odyssey Infra-red Imaging System (LI-COR^® Bioscience, Lincoln, Nebraska USA). Anti α-tubulin antibody (1:2000, Sigma-Aldrich) was used to assess equal amount of protein loaded in each lane.

The following antibodies were used: mouse anti- FLAG M2 (Sigma-Aldrich), rabbit anti-HA tag (Abcam), rabbit anti-E-cadherin (Cell Signaling Technology), mouse anti-E-cadherin (DAKO), rabbit anti-ZEB1 (Cell Signaling Technology), rabbit anti-Snail1 (Cell Signaling Technology), rabbit anti-Twist1 (BIORAD), mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-TCF4 (Santa Cruz Biotechnology), mouse anti-β-catenin (Sigma-Aldrich), rabbit anti-phospho-STAT3 (Abcam), rabbit anti-STAT3 (Abcam), rabbit anti-galectin-3 (Abcam), goat anti-Trop-2 (R&D), and HRP-conjugated mouse anti-FLAG M2 antibodies (Sigma-Aldrich). Rabbit anti-phospho-Trop-2 antibodies were prepared as described previously (23 (link)).