Anti β3

The human prostate cancer cell lines C4-2, 22RV1 and DU145 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The PEP06 and 33 polypeptide were synthesized by Ningbo Kangbei Biochemical Co., Ltd. (Ningbo, China). RPMI 1640 medium was purchased from Thermo Fischer Scientific Inc.(Waltham, MA, USA) FBS, penicillin and streptomycin were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Trypsin was purchased from Invitrogen (Carlsbad, CA, USA). Matrigel basement membrane matrix was purchased from Seebio Biotechnology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from VWR International Co. Transwell cell inserts were purchased from Wuxi NEST Biotechnology Co., Ltd. (Wuxi, China) Rabbit anti-αv, anti-β3, anti-α6, anti-β1, anti-MMP2, anti-E-cadherin, anti-β-catenin, anti-vimentin, anti-MMP9, anti-AKT and anti-P-AKT monoclonal antibodies were purchased from Abcam Trading Co., Ltd. (Shanghai, China).

Abs against P-Axl(Y702) (rabbit), P-AKT(S473) (rabbit), anti-Mer (mouse), P-src (Y416), and anti-P-Tyrosine (P-Tyr-100) (mouse) were obtained from Cell Signaling Technology (New England BioLabs, Beverly, MA, USA); anti-Axl (goat), anti-FAK (rabbit), anti-Tyro-3 (goat), and anti-β1 and anti-p130Cas (mouse) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-α-tubulin (mouse) from Thermo Scientific (Fremont, CA, USA); anti-β-actin (rabbit) from Sigma-Aldrich (Saint Louis, MO, USA); anti-β3 from Abcam (Cambridge, UK); and anti-β4 from Millipore (Merck Millipore, Oxford, UK).
Alexa Fluor 488 phalloidin was obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Human recombinant Gas6 and Axl-Fc were from R&D systems, Inc. (Minneapolis, MN, USA), as was the ELISA for Gas6 dosage. FN was from Sigma-Aldrich. The Taqman Gene Expression Assays were from Applied Biosystems (Foster City, CA USA). Puromicine and Lipofectamine 2000 were from Invitrogen. R428, the Axl inhibitor, was from Rigel (South San Francisco, CA, USA); EHT1864, the Rac inhibitor, was from Tocris (Minneapolis, MN, USA).

Protein samples in reducing sample buffer were boiled for 5 minutes and applied to 4‐12% NuPage Gels and separated by electrophoresis using the Xcell SureLock system (Invitrogen, Paisley, UK) under reducing conditions. Proteins were then transferred on to nitrocellulose membrane (Millipore, Bedford, UK) at 40 V overnight at 4°C using a Mini Protean II system (Bio‐Rad, Hemel Hempstead, UK). Following transfer, the PVDF was blocked (5% nonfat dry powdered milk, 0.1% Tween 20 in TBS) for 1 h and primary antibody was then added (1:1000 dilution) and incubated for 2 h at room temperature. Anti‐human GPVI was a kind gift from Dr. P. Smethurst, and anti‐β3 was obtained from Abcam, Cambridge, UK. Following washes with TBST, the membrane was incubated with HRP conjugated secondary antibody (1:10000 dilution/TBST) for 1 h at 24°C. The PVDF was developed using a chemiluminescent substrate (GE Healthcare, Amersham, Bucks, UK).