Mmp 2

Equal amounts of protein (30 μg) were resolved by SDS-PAGE and then electrophoretically transferred onto PVDF membranes (Millipore, Bedford, MA). After blocked in 5% non-fat milk 1 h at room temperature, the membranes were incubated with appropriately diluted primary antibodies overnight at 4 °C. After washed twice with TBST, the blotted membranes were incubated with HRP-conjugated secondary antibody at 1:20000 dilutions for 1 h at room temperature. The membranes were visualized by the enhanced Phototope TM-HRP Detection Kit and exposed to Kodak medical X-ray processor (Carestream Health, USA). The primary antibodies are as followings: SPAG5, 1:500 (Sigma-Aldrich), CEP55 (1:1000, #81693, Cell Signaling Technology), AKT (1:1000, #2920, Cell Signaling Technology), p-AKT at Ser473 (1:1000, #4046, Cell Signaling Technology), ERK1/2 (1:1000, #4695, Cell Signaling Technology), p-ERK1/2 at Thr202/Tyr204 (1:1000, #4370, Cell Signaling Technology), E-Cadherin, β-Catenin, N-Cadherin, Vimentin, Fibronectin and MMP-2 (Epitomics, Burlingame, CA) and β-actin (1:1000, #3700, Cell Signaling Technology).

Phenylmethylsulfonyl fluorides (PMSF), bovine serum albumin (BSA), DL-Glyceraldehyde, and PD98059 were purchased from Sigma (St. Louis, MO, USA). DMEM media, fetal bovine serum (FBS), penicillin, streptomycin, Hanks Balanced Salt Solution (HBSS), trypan blue, and Lipofectamine RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). GAPDH (Cat No.: MAB374) was purchased from Chemicon (Temecula, CA, USA). ERK (Cat No.: SC-94), p-ERK (Cat No.: SC-7383), RAGE (Cat No.: SC-94), and RAGE siRNA (Cat No.: SC-36374) were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). MMP2 (Cat No.: 2763-S) and MMP9 (Cat No.: 2551-S) were purchased from Epitomics (Burlingame, CA, USA). Nitrocellulose membranes were purchased from PALL corp. (Ann Arbor, MI, USA). Enhanced chemiluminescence (ECL) was purchased from Millipore (Billerica, MA, USA). Wst-1 kit was purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA). Culture-Insert was purchased from ibidi (Verona, WI, USA).

BBR was obtained from Sigma and was dissolved at a concentration of 100 mM in dimethyl sulfoxide (DMSO, Sigma-Aldrich) as a stock solution (stored at -20°C). It was then diluted to working concentrations with cell culture medium. The maximum final concentration of DMSO was less than 0.1% for each treatment, and was also used in controls. Recombinant human TGF-β1 was purchased from Peprotech. Rabbit monoclonal antibodies against human E-cadherin, Slug, Snail, Vimentin, MMP-2 and MMP-9 were purchased from Epitomics. P-Smad2/3(Ser423/425) and Smad 2/3 were purchased from Cell Signaling. Matrigel (BD Biosciences) and 24-well transwells (Corning) were used.

Animals (n = 3 for each group) were anesthetized and transcardially perfused with saline. The samples of brain metastasis were rapidly isolated and stored at −80°C. The expressions of urokinase-type plasminogen activator (uPA), osteopontin (OPN), matrix metalloproteinase-2 (MMP-2) and CXCR4 in the location with brain metastasis were determined using western blot analysis. The same location in the brains of normal mice was also analyzed as a negative group. The following antibodies were used: anti-urokinase-type plasminogen activator (uPA) (Santa Cruz Biotechnology, USA), anti-OPN (Abcam, USA), anti-matrix metalloproteinase-2 (MMP-2, Epitomics, USA), and anti-CXCR4 (Abcam, USA).