Anti n cadherin

Anti-ARHGEF5 was generated in rabbits via immunization with GST-mouse or human ARHGEF5 (amino acids 2–204) and affinity purified using a maltose-binding protein-tagged antigen. Anti-Src-pY418, anti-GFP, anti-FAK-pY397, SD208, Alexa Fluor 488 phalloidin, Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G, horse radish peroxidase-conjugated goat anti-rabbit immunoglobulin G, anti-mouse immunoglobulin G, anti-occludin and anti-cortactin-pY421 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH, anti-Fyn, anti-Lyn and anti-vimentin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-v-Src, anti-cortactin (4F11) and anti-phosphotyrosine (4G10) were from Millipore (Billerica, MA, USA). Anti-FLAG (M2) and anti-β-tubulin were from Sigma-Aldrich (St Louis, MO, USA). Anti-E-cadherin, anti-N-cadherin and anti-FAK were from BD Transduction Laboratories (Lexington, KY, USA). anti-Smad2-pS465/467, anti-Smad2, anti-MLC2-pT18/S19, anti-MLC2, anti-Akt and anti-Akt-pS473 were from Cell Signaling Technology Inc. (Beverly, MA, USA). TGF-β1 and TNF-α were from PeproTech (Rocky Hill, NJ, USA). The Akt inhibitor triciribine was from Selleckchem (Houston, TX, USA).

Total cellular proteins were extracted using lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100 and protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Extracted proteins were quantified using the BCA protein assay kit. 30 mg of total proteins were separated using 12% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA). The following antibodies were used in the current study: anti-AUF1 (EMD Millipore, 07–260), anti-ZEB1 (AREB6) (Abcam, ab155249), anti-Beclin1 (MBL, PD017), anti-E-cadherin (Cell Signaling, 3195), anti-N-cadherin (BD Transduction Laboratories, 610920), anti-Fibronectin (Abcam, ab2413) and anti-GAPDH (EMD Millipore, ABS16).

Cells were lysed in protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na₂P₂O₇, 1 mM Na₃VO₄, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM PMSF, and protease inhibitor cocktail). Lysates containing equal amounts of proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blots were blocked with a 5% skim milk solution and incubated with the following antibodies: anti-Dicer, anti-cyclin A, anti-cyclin D1, anti-cyclin E, anti-p21^WAF1/CIP1, anti-EZH2,(Cell Signaling Technology, Danvers, MA), anti-HDAC2, anti-GAPDH and anti-CDK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-N-cadherin, anti-E-cadherin, anti-vimentin and anti-fibronectin (BD Transduction, San Jose, CA), anti-DNMT1 (Abcam, Cambridge, MA) and anti-H3K27me3 (Millipore, Billerica, MA). The Immobilon™ Western blot detection system (Millipore, Billerica MA) was used to detect bound antibodies. The intensities of the Western blot bands were quantified using LAS 3000 (Fuji Photo Film Co., Japan).

Whole cell lysates were obtained using the M-Per Mammalian Protein Extraction Reagent (Pierce Biotechnology, Woburn, MA). Nuclear protein extracts were prepared using a Nuclear Extraction Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Total proteins (40 μg) and nuclear proteins (10 μg) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Antigen-antibody complexes were detected using the enhanced chemiluminescence (ECL) blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The following antibodies were used for analysis: anti-EZH2 (5246, Cell Signaling, Danvers, MA), anti-PARP (9542, Cell Signaling, Danvers, MA), rabbit polyclonal anti-E-cadherin (A01589, GenScript, Edison, NJ), mouse monoclonal anti-N-cadherin (BD, Transduction, San Jose, CA), rabbit polyclonal anti-Vimentin (A01189, GenScript, Edison, NJ). Anti-MCL-1 (sc-819), anti-FOS (sc-52), anti-p21 (sc-397), anti-Bax (sc-493), anti-β-catenin (sc-1496), and anti-GAPDH (sc-47724) and anti-lamin B1 (sc-20682) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA). GAPDH (whole cell lysate) and lamin B (nuclear protein) were blotted to show equal protein loading, respectively.

Western blotting was performed as previous described [15 (link)]. The primary anti-MAP17 antibodies (#ab156014, 1:1000) were purchased from Abcam (Cambridge, UK) and used for the Western blotting and immunohistochemical (IHC) analyses. The anti-E-cadherin (#610,182, 1:1000) and anti-N-cadherin (#610,920, 1:1000) antibodies were purchased from BD Transduction Laboratories (San Jose, CA, USA). The anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #2118, 1:10,000) antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti-vimentin antibodies (#sc-32322, 1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

The following antibodies were used in our study: anti-ERK1/2 (Cat# 61-7400; Thermo Fisher Scientific, Waltham, MA, USA), anti-phospho-ERK1/2 (9101; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-p70 S6 kinase (Cat# 9206; Cell Signaling Technology), anti-phospho (Ser/Thr) AKT substrate (Cat# 9611; Cell Signaling Technology), anti-phospho-p85 (Cat# 9206; Cell Signaling Technology), anti-Smad4 (Cat# sc-7966; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin (Cat# 610181; BD Transduction Laboratories, BD Biosciences, San Jose, CA, USA), anti-N-cadherin (Cat# 610921; BD Transduction Laboratories), anti-Vimentin (Cat# sc-32322; Santa Cruz), anti-Snail1 (Cat# C15D3; Cell Signaling Technology), anti-Enolase1 (Cat# 3810; Cell Signaling Technology), anti-GAPDH (Cat# sc-47724; Santa Cruz), anti-ALDOA (Cat# 11217-1-AP; Proteintech, Proteintech Europe Ltd, Manchester, UK), anti-Pyruvate kinase (Cat# 3106; Cell Signaling Technology), anti-Lamin A/C (Cat# sc-376248; Santa Cruz), anti-g-Catenin (Cat# A0963; ABclonal, Woburn, MA, USA), anti-Plakophilin (Cat# sc-33636; Santa Cruz), anti-β-Actin (Cat# A1978; Sigma-Aldrich, St Louis, MO, USA). GSK690693, U0126 and SB-203580 were purchased from Selleck Chemicals (Houston, TX, USA). TGF-β was purchased from PeproTech (London, UK).