Anti fak

Cells were washed with PBS and lysed with n-octyl-β-D-glucoside (ODG) buffer [20 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1 mM EDTA, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 1% Nonidet P-40, 5% glycerol, 2% ODG, and a protease inhibitor cocktail], and immunoblotting was carried out as described previously [26] (link). The following primary antibodies were used: anti-Src (Ab-1; Calbiochem), anti-Src pY418 (Invitrogen), anti-phosphotyrosine (4G10; Millipore), anti-cortactin (Sigma), anti-cortactin pY421 (Sigma), anti-FAK (Santa Cruz), anti-FAK pY576 (Cell Signaling), anti-ERK pT202/Y204 (Cell Signaling), anti-ERK (Cell Signaling), anti-GAPDH (Sigma), anti-Flottilin-1 (BD) and anti-Caveolin-1 (BD). Anti-Cbp and anti-Cbp pY314 were prepared as described previously [19] (link). GAPDH was used as a loading control. Horseradish peroxidase (HRP)–conjugated anti-mouse or anti-rabbit IgG (Zymed) was used as the secondary antibody. All blots were visualized and quantitated using a LAS-4000 luminescent image analyzer (GE Healthcare).

HepG2 cells were made quiescent by a 3 h starvation in DMEM without FCS and stimulated for 15 min with indicated doses of BMP4 pre-incubated in the absence or the presence of the indicated doses of recombinant gremlin. Alternatively, HUVECs were made quiescent by a 20 h starvation and stimulated with the indicated doses of gremlin for 10 min. After the treatment, cells were lysed in 50 mM Tris–HCl buffer (pH 7.4) containing 1.0% Triton-X 100, 0.1% BriJ, 1.0 mM sodium orthovanadate and protease inhibitor cocktail. An amount of 100 µg of proteins was subjected to SDS-PAGE followed by WB by using anti-phospho-SMAD1/5/8 (Cell Signaling, Danvers, MA, USA), anti-phospho-VEGFR2 (Y1175) (ThermoFisher Scientific, Waltham, MA, USA) or anti-FAK (Santa Cruz, Dallas, TX, USA) antibodies.

Assays were set up 48 or 72 hours after transfection. EGF treatment (30 uM, 15 minutes - Millipore) was performed on serum-starved cells, whereas all other experiments were conducted in the presence of serum. Each assay was independently repeated three times with similar results; the blots with the most equalized loading as judged by β-actin labelling are shown.
Cells were washed in PBS (Phosphate buffered saline) and lysed in lysis buffer (2% SDS, 100 mM Tris-HCl, pH 7.4). Cell protein extracts were incubated at 95°C for 5 minutes, sonicated and clarified by centrifugation at 10,000 g for 15 min. Protein concentration was determined by Bradford method [76] (link). Proteins were resolved by SDS-PAGE and analysed by Western blotting. The following antibodies were used for immunoblotting: anti-β-actin (1∶10000, Abcam), anti-FAK (1∶1000, C-20, Santa Cruz Biotechnology), anti-phosphorylated (T202/Y204)-MAPK (pERK1/2) (1∶200, Cell signalling), anti-ERK1/2 (1∶10000, Promega), anti-EGFR (1∶2000, BD Transduction Laboratories). The Nitrocellulose membranes were incubated with secondary antibodies IRDye 680RD anti-mouse (1∶10000, LI-COR) and IRDye 800CW anti-rabbit (1∶10000, LI-COR) imaged with Odyssey Imager (LI-COR Biosciences) and analysed with Image Studio Lite software.

PMA, ATP, nigericin, PF-431396, 4′,6-diamidino-2-phenylindole (DAPI), and the anti-Flag M2 antibody were purchased from Sigma. R406, PF-562271, and Z-VAD-FMK were purchased from Selleckchem. Anti-Pyk2¸ anti-Syk, anti-p-Pyk2, anti-p-FAK, anti-p-Syk, and anti-GAPDH were purchased from Cell Signaling. Anti-FAK, anti-ASC, anti-caspase-1, and anti-IL-1β were purchased from Santa Cruz. Anti-Ly6G-PE and anti-CD11b-APC were purchased from BD Bioscience. MSU, PF-573228, anti-NLRP3, anti-mCherry, anti-F4/80-FITC, and Alexa Fluor 488–conjugated goat anti-rabbit IgG were purchased from InvivoGen, Tocris Bioscience, AdipoGen, Abcam, eBioscience, and Invitrogen, respectively. Plasmids encoding His-tagged wild-type and mutant (Y146F) ASC were generated by ligation of amplified DNA fragments to NdeI/XhoI-treated pET15b (Novagen, EMD Millipore, Darmstadt, Germany). The His-tagged mutant ASC (Y146F) was constructed using a QuikChange Site-Directed Mutagenesis kit (Stratagene) with forward primer 5′- GACGG ATGAG CAGTT CCAGG CAGTG CGGGC -3′ and reverse primer 5′- GCCCG CACTG CCTGG AACTG CTCAT CCGTC -3′. The mutant sequences are underlined. pENTER-Pyk2-Flag was purchased from ViGene Biosciences. The expression vectors for ASC-Flag and ASC were constructed using the pLKO_AS2 vector (RNAi Core, Taiwan), and that for Flag-NLRP3 was constructed using the pFLAG-CMV2 vector (Sigma).

Anti-M13 fused to HRP (phage display library kit, New England Biolabs), anti-CAS (BD Transduction Laboratories, mouse monoclonal clone 21), anti-GST (G7781, Sigma, rabbit polyclonal), anti-FLAG (Sigma, mouse monoclonal clone M2), anti-FAK (C–20, Santa Cruz Biotechnology, rabbit), anti v-Src (Ab–1, Calibiochem, mouse monoclonal clone 327), anti-Vinculin (Sigma, mouse monoclonal clone V284), anti-CAS pY165 (#4015, Cell Signaling Technology, rabbit polyclonal), anti-Src pY418 (#2101, Cell Signaling Technology, rabbit polyclonal), anti-Actin (C–11, Santa Cruz, goat polyclonal), and secondary antibody/ies fused to HRP (Abcam) were used as purchased. Anti-CAS Y12 antibody was prepared as previously described^{20 (link)}.