Recombinant human tgf β1 protein

Anti-VE-cadherin (F-8, 1 µg/ml) and DAPI were from Santa Cruz Biotechnology. Anti-E-cadherin (ab1416, 1 µg/ml), anti-Ki67 (ab15580, 0.25 µg/ml), and anti-β tubulin (ab6046, 0.25 µg/ml) were from Abcam. Anti-GM130 (clone 35, 2 µg/ml) was from BD Biosciences. Anti-VEGFA (VG-1, 2 µg/ml), anti-ZO-1 (40-2200, 1 µg/ml), rhodamine phalloidin (1 µg/ml), 70 kDa FITC-dextran and AlexaFluor 647 conjugated goat secondary antibodies were from Life Technologies. Anti-α6 integrin (MA6, 1 µg/ml) was from Millipore. Anti-HA (6E2, 0.5 µg/ml), anti-GFP (D5.1, 0.5 µg/ml), and anti-GAPDH (D16H11, 0.25 µg/ml) were from Cell Signaling Technologies. HRP-conjugated donkey anti-mouse and rabbit IgG secondary antibodies (1: 0.25 µg/ml) were purchased from Fitzgerald. Recombinant human IL-6 protein (7270-IL, 200 ng/ml), recombinant human TGFβ1 protein (240-B, 5 ng/ml), recombinant human FGF2 protein 2 (33-FB, 3 nM), anti-IL-6 (6708, 1 µg/ml), anti-IL-6Rα (MAB227, 0.5 µg/ml), and Proteome Profiler Human Cytokine Array Kit were purchased from R&D Systems. Semaxanib was purchased from Selleckchem.

Polyclonal anti-Shc1 antibody was obtained from Thermo Scientific Pierce (Rockford, IL), and polyclonal anti-MADH7 (Smad7) antibody was purchased from Abcam Inc. (Cambridge, MA). Anti-HDAC2 was purchased from Millipore (Billerica, MA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): anti-pSma2 (linker and COOH specific phosphorylation), anti-Smad2, anti-pSmad3, anti-Smad3, anti-pAKT, anti-pERK, anti-BIM, anti-PARP, and anti-GAPDH. Recombinant human TGFβ-1 protein was purchased from R&D Systems (Minneapolis, MN) and reconstituted in 4 mM HCL and 1 mg/mL bovine serum albumin solution. Dasatinib and erlotinib were obtained from ChemieTek (Indianapolis, IN) and diluted in DMSO. LY-364947 was purchase from Sigma-Aldrich (St. Louis, MO). AZD0530 was obtained from AstraZeneca (London, UK).

Recombinant human TGF-β1 protein was from R&D systems (Abingdon, UK). Fenoterol was purchased from Boehringer Ingelheim (Ingelheim, Germany). Rolipram, cilostamide, and bovine serum albumin (BSA) were from Sigma-Aldrich (St-Louis, MO, USA). Forskolin was from Tocris Bioscience (Bristol, UK). InCELLect™ AKAP St-Ht31 inhibitor peptide was purchased from Promega (Leiden, the Netherlands). The transfect reagent lipofectamine RNAiMax was purchased from Invitrogen (Bleiswijk, the Netherlands). Control siRNA, Ezrin siRNA, AKAP95 siRNA, and Yotiao siRNA were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). All other chemicals were of analytical grade. Table 1 lists the origin and dilution of the antibodies used.

The mouse anti-paxillin antibody was purchased from BD Biosciences (610052) and the corresponding secondary Alexa488 anti-mouse antibody from ThermoFisher Scientific (A-11029). The rabbit anti-fibronectin antibody was obtained from Sigma (F3648) and the corresponding secondary Alexa647 anti-rabbit antibody from ThermoFisher Scientific (A-21245). Alexa Fluor 568-coupled phalloidin was from ThermoFisher Scientific (A12380). Rat anti-E-cadherin antibody was obtained from ThermoFisher Scientific (13-1900), rabbit anti-ZO1 antibody from ThermoFisher Scientific (61-7300), mouse anti-vimentin antibody from Sigma (V2258), mouse anti-SMAD2/3 from BD Biosciences (610842), rabbit anti-p-SMAD3 from Cell Signaling (p-Ser423/425; 9520) and mouse anti-GAPDH from Sigma (G8795). For quantitative immunoblot analysis, secondary antibodies from LI-COR Biosciences, IRDye 680RD Goat anti-Mouse (926-68070) and IRDye 800CW Goat anti-Rabbit (926-32211) were used. DAPI was acquired from Sigma (D9542), recombinant human TGFβ1 protein from R & D Systems (240B-0-10), 16% paraformaldehyd (PFA) from Electron Microscopy Services (15710-S), fatty-acid free BSA from Calbiochem (126575), Trypsin/EDTA from Sigma (T4174), PBS from Gibco (14200-067), and Triton X-100 from Sigma (X-100).

Cells were growth arrested in serum-free media for 24 h prior to stimulation in the presence or absence of inhibitors. After serum starvation, cells were stimulated with either 10 μg/ml recombinant human galectin-3 protein (R&D Systems) or 2 ng/ml recombinant human TGF-β1 protein (R&D Systems) for 2 h or 50 μM LPA (Sigma-Aldrich) for 4 h.
When used, inhibitors were applied in serum-free media for 20 min prior to stimulation. All of the small molecule glycomimetics utilized in this study, GB0139 (formerly TD139), GB1107, GB1211, or GB0149 were synthesized by the Galecto Biotech AB Medicinal Chemistry Department (Table S1) and used at a concentration range of 0.1 to 10 μM. The ALK5/TGFβRI inhibitor SB-525334 (Sigma-Aldrich) (62 (link), 63 (link)) was used at a concentration of 50 μM. The β1 integrin inhibitor NOTT199SS (gifted) was used at a concentration range of 0.1 to 100 nM. All inhibitors were dissolved in 100% dimethyl sulfoxide (DMSO), and all cells, including untreated controls, were exposed to a DMSO concentration equivalent to that used in the highest inhibitor concentration in each experiment.

Costunolide (≥98%) was obtained from Nanjing Plant Origin Bio-Technology Co., Ltd. (Nanjing, China). Methacrylic acid copolymer Type A was obtained from Rohm (Germany). Tetraethyl orthosilicate (TEOS) and hexadecyl trimethyl ammonium bromide (CTAB) were purchased from Aladdin (Shanghai, China). 2,2′-Azobis (2-methylpropionamide) dihydrochloride (AIBA, ≥99%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). L-lysine was purchased from Rhawn Reagent (Shanghai, China). Recombinant human TGF-β1 protein was obtained from R&D Systems (R&D, USA). Antibodies for α-SMA were obtained from Abcam (Abcam, UK). Antibodies for TGF-β1, MMP2, COL1A1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HRP-conjugated secondary antibodies against mouse or rabbit IgG were purchased from Proteintech (Wuhan, China). A rapid block buffer was obtained from New Cell & Molecular Biotech Co., Ltd (Suzhou, China). All other reagents were of analytical grade and were obtained from commercial sources.