Anti fibronectin

Assays were performed as previously described, with modification [40 (link), 69 (link)]. The antibodies were obtained from the following companies: anti-HA(Y-11), anti-c-Src, and anti-Myc (9E10) were from Santa Cruz; anti-V5 was from Invitrogen; anti-TrkB and anti-SOCS3 were from Abcam; anti-phospho-c-Src, anti-Twist-1, anti-STAT3, anti-phospho-STAT3, anti-JAK2, and anti-phospho-JAK2 were from Cell Signaling Technology; and anti-E-cadherin, anti-fibronectin, anti-N-cadherin, anti-α-catenin, and anti-β-catenin were from BD Transduction.

Cell lysates were collected in RIPA buffer. Proteins were separated on SDS-PAGE and subsequently transferred from gel onto PVDF membrane (Pall, Port Washington, NY). Membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline plus 0.01% Tween-20 (TBS-T). Smad2/3 antibody sampler kit (Cell Signaling, #12747), anti-fibronectin (BD, 610077), anti-E-cadherin (BD, 610182), anti-HIF-1α (BD, 610959), anti-ENOSF1 (GeneTex, GTX119464), anti-RAB6A (GeneTex, GTX110646), anti-phopho-TAB2 (Cell Signaling, #8155), anti-TAB2 (Cell Signaling, #3745) and anti-β-actin (Sigma, A5441) antibodies, were used to detect p-Smad2, Smad2, p-Smad3, Smad3, Smad4, fibronectin, E-cadherin, HIF-1α, ENOSF1, RAB6A, p-TAB2, TAB2 and β-actin, respectively, with incubation of membrane at 4 °C overnight. After washing three times with TBS-T, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Signal was detected with an enhanced chemiluminescence detection kit (Perkin Elmer Life Sciences, Boston, MA).

Cells were lysed on ice in buffer containing 50mM HEPES, pH7.4, 80mM NaCl, 5mM MgCl₂, 10mM EDTA, 5mM sodium pyrophosphate*10H₂O, 1% TritonX-100, and Protease Inhibitor Cocktail (Sigma-Aldrich). 30 μg of total protein from each sample was resolved on Novex 4-20% Tris-Glycine Mini Protein Gels and transferred onto nitrocellulose membranes. The blots were probed with the appropriate antibodies: anti-ubiquitin (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich), anti-20S/β1 subunit (Santa Cruz Biotechnology, Inc.), anti-20S/β2 subunit (Santa Cruz Biotechnology, Inc.), anti-20S/β5 subunit (Santa Cruz Biotechnology, Inc.), anti-E-cadherin (BD Biosciences), anti-fibronectin (BD Biosciences), anti-vimentin (Cell Signaling Technology), anti-phospho-Smad2 (Ser465/467) (Cell Signaling Technology), or anti-Smad2/3 (Cell Signaling Technology). Signals were detected using Odyssey infrared imaging system (LI-Cor Biosciences).

Following growth on coverslips for 2 days and treatment with DMSO, β2 inhibitor, β5 inhibitor or TGF-β1 with or without anti-TGF-β1 antibodies, immunofluorescence of HMLEs and MCF10A was performed as previously described [44 (link)] using anti-E-cadherin (BD Biosciences), anti-fibronectin (BD Biosciences), anti-vimentin (Cell Signaling Technology), or anti-Smad4 (B-8) (Santa Cruz Biotechnology) followed by anti-rabbit Alexa Fluor 555 or anti-mouse Alexa Fluor 488 (Life Technologies) antibodies. DAPI (Life Technologies) was used to detect DNA. Acquisition of image stacks was performed as previously described [44 (link)] using a FV1000 laser scanning confocal microscope (Olympus). Fluorescence within the nucleus or cytoplasm was quantified using ImageJ software.

These reagents were purchased from the indicated sources, including cisplatin (FUJIFILM Wako Pure Chemical #039-20093, Tokyo, Japan), TGF-β1 (R&D Systems #240-B, Minneapolis, MN), SB431542 (Sigma-Aldrich #S4317, St. Louis, MO), and TβRI kinase inhibitor II (TK-II, RepSox) (Merck Millipore #616452, Billerica, MA). The following antibodies were obtained from the indicated sources, including anti-E-cadherin (#610181), anti-N-cadherin (#610920), and anti-fibronectin (#610077) that were purchased from BD Biosciences (San Jose, CA). The anti-vimentin (#V6389) antibody was purchased from Sigma-Aldrich. Anti-slug (#9585) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-Smad3C (#28031) antibody was purchased from IBL (Gumma, Japan). Furthermore, anti-Smad2/3 (sc-8332) and anti-β-actin (sc-69879) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).