Anti β1 integrin

Immulon 2HB 96-well plate (ThermoFisher Scientific) was coated with LME8 (LM111E8, LM211E8, LM411E8, or LM511E8) at 30 nM. The substrate-coated plates were incubated at 4 °C overnight. After blocking with 1% BSA for 1 hour at 37 °C, sorted Venus positive OPCs in 0.1% BSA/DMEM were plated on coated wells at 1.0–2.0 × 10⁴ cells per well and incubated for 1 hour at 37 °C. The attached cells were stained with 0.2% crystal violet aqueous solution in 20% methanol for 10 minutes at room temperature and counted. In the inhibitory assay, the cell suspension was pre-incubated at 37 °C for 15 minutes with 10 μg/ml of the inhibitory antibody. Antibodies were as follows: anti-α3 integrin (clone name: P1B5; Merck Millipore), anti-α6 integrin (clone name: NKI-GoH3; Merck Millipore), anti-β1 integrin (clone name: 9EG7; BD Biosciences), anti-β4 integrin (clone name: ASC-9; Merck Millipore) and normal rat IgG (Santa Crus Biotechnology) as a control.

Anti-p53, anti-p21^waf1, anti-cyclin D1, and anti-bcl2 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-HNF-1β, anti-GSK-3β, anti-Rb, anti-p27^kip1, anti-XIAP, anti-bax, and anti-integrin β1 antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-ARID1A, anti-cyclin B1, and anti-MDM2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail, anti-Akt, anti-phospho(p) Akt Serine473, anti-pGSK-3β Serine9, anti-pRb Serine807/811, anti-cleaved caspase-3, and anti-integrin β3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (FN), anti-E-cadherin, and anti-β-actin antibodies were obtained from Abcam (Cambridge, MA, USA), Takara (Shiga, Japan), and Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. Anti-cyclin A2 and anti-integrin β2 antibodies were from Novocastra (Newcastle, UK) and Merck KGaA (Darmstadt, Germany), respectively. FN (catalog number #F2006) and cisplatin (CDDP: #479306) were purchased from Sigma-Aldrich Chemicals.

The rabbit polyclonal anti-claudin-1 (Cat. 51–9000), −2 (Cat. 516100), −3 (Cat. 341700), −4 (Cat. 364800), ZO-1 (Cat. 61–7300), and mouse monoclonal anti-myc (Cat. 46–0603) antibodies were from Invitrogen (Carlsbad, CA). The rabbit polyclonal anti-claudin-7 antibody (Cat. 18875) was obtained from Immuno-Biological Laboratories (Japan). The anti-E-cadherin antibody (Cat. 610182, clone: 36) was purchased from BD Biosciences (San Jose, CA). The rabbit polyclonal anti-phospho-Bcl-2 (Cat. A7025, Ser56) was from Assay Biotechnology (San Francisco, CA). The rabbit polyclonal anti-phospho-FAK (Cat. 3283S) and anti-phospho-ERK1/2 (Cat. 9101S) were from Cell Signaling Technology (Beverly, MA). The mouse monoclonal and the goat polyclonal anti-integrin β1 antibodies were purchased from BD Biosciences (Cat. 610467) and Santa Cruz Biotechnology (Cat. sc-6622, Santa Cruz, CA), respectively. The mouse anti-integrin β1 adhesion-blocking antibody was from Chemicon (Cat. MAB22253Z, Millipore, MA).

Cells were fixed as previously described [47 (link)], and incubated with anti-integrin β1, anti-integrin β4, anti-paxillin, anti-E-cadherin (BD Biosciences, 610467, 611232, 6100052, and 610181, respectively), anti-integrin β3, anti-Giantin (Abcam Ab7167 and 24586 respectively), and anti-α-catenin (B52975; Calbiochem), for 1 h, and then incubated with the corresponding secondary antibodies and FITC-phalloidin (Invitrogen) at 1 μg/mL for 30 min.

pSM2 vector expressing small hairpin RNA against Canis lupus familiaris DDR1 was purchased from GenDiscovery Biotechnology35 (link). pcDNA3 expressing vector-encoding human E-cadherin (HECD) was obtained as a gift from Dr Barry M. Gumbiner (Memorial Sloan-Kettering Cancer Center, New York, USA). The p221-Ecadh-mEosFP plasmid provided by Dr. Thomas Lecuit was reconstructed as previously described35 (link).
Polyclonal anti-DDR1, anti-α-catenin, and monoclonal anti-c-Src antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Monoclonal anti-E-cadherin, anti-β-catenin, and anti-integrin β1 antibodies were purchased from BD Biosciences (San Jose, CA, USA). Monoclonal anti-β-actin antibody was purchased from Novus Biologicals (Littleton, CO, USA). Polyclonal anti-EEA-1 and anti-clathrin heavy chain antibodies were purchased from Abcam (Cambridge, MA, USA). Monoclonal anti-E-cadherin antibody (rat IgG1 isotype) was purchased from Sigma-Aldrich (St Louis, MO, USA). Polyclonal anti-Src pY418 antibody was purchased from ThermoFisher Sci-Invitrogen (Grand Island, NY, USA). anti-integrin β1 blocking antibody, 4B4, was purchased from Beckman Coulter (Indianapolis, IN, USA). Small interfering RNA (siRNA) specifically binding to a human DDR1 mRNA sequence (ONTARGETplus siRNA pool) was purchased from GE Dharmacon (Lafayette, CO, USA).