Anti integrin α4

Protein was extracted from the cells and quantified as described previously [18, 19] . Equal amounts of protein were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk in TTBS (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.5% Tween-20) for 40 min at room temperature and incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used: anti-LBH, anti-ZEB1, anti-ZEB2, anti-SNAIL, and anti-SLUG (Abcam); anti-integrin-α1, anti-integrin-α2, anti-integrin-α4, anti-integrin-αv, anti-integrin-β1, anti-integrin-β3, anti-integrin-β4, anti-integrin-β5, anti-E-cadherin, anti-vimentin, and anti-actin (Cell Signaling Technology, Boston, MA, USA). The membranes were washed in TTBS and incubated with secondary antibodies for 40 min. After extensive washing, the membranes were visualized using the enhanced chemiluminescence reagent. Final images were analyzed using ImageJ software.

Immunoblotting procedures were as described above. Blots were incubated with rabbit, mouse or goat polyclonal primary antibodies (Santa Cruz Biotechnology, except when differently stated) as follows: anticollagen type VI alpha 1 chain (COL6A1), 1:200; anti-tenascin C (TNC), 1:1000; anti-integrin β7, 1:500; anti-integrin α4, 1:500; anti-focal adhesion kinase (FAK; Cell Signaling Technology), 1:1000; anti-Rhoassociated protein kinase 1 (ROCK1; Cell Signaling Technology), 1:1000; anti-vimentin, 1:1000; antidetyrosinated α-tubulin (de Tyr α-Tubulin; Abcam, Cambridge, UK), 1:500; anti-vinculin, 1:500; anti-citrate 12 synthase, 1:500; anti-fatty acid synthase (FASN), 1:100; anti-peroxisome proliferator-activated receptors gamma (pparγ), 1:500; anti-peroxisome proliferator-activated receptors alpha (pparα), 1:500; anti-heat shock cognate 71 kDa protein (HSC70), 1:1000; anti-myosin heavy chain 9 (MYH9), 1:500; anti-major vault protein (MVP); 1:2000; anti-ubiquitin-binding protein p62 (p62; Sigma Aldrich), 1:1000; anti-light chain 3B (LC3B; Cell Signaling Technology), 1:500; anti-Bcl-2-like protein 4 (BAX), 1:1000; anti-beclin1, 1:1000; anti-B-cell lymphoma-extra large (Bcl-xL; Cell Signaling Technology), 1:1000; anti-BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) 1:1000; B-cell leukemia/lymphoma 2 (Bcl-2; Cell Signaling Technology).