Anti fak

Paxillin, focal adhesion kinase (FAK) and vinculin expression in fascin-silenced HSC-3 cells and controls was assessed by western blot, using the following antibodies: anti-paxillin (diluted 1:500; Abcam Inc, USA), anti-phospho paxillin (diluted 1:500; Cell Signaling, USA), anti-FAK (diluted 1:500; Millipore, USA), anti-phospho-FAK (diluted 1:500; Cell Signalling, USA), anti-vinculin (diluted 1:500; Sigma-Aldrich, USA), anti-GAPDH (diluted 1:500; Sigma-Aldrich, USA). Western blot with phospho-fascin (1:50, clone FP2661; ECM Bioscience, USA) was also performed.
To assess the effects of epidermal growth factor (EGF) stimuli on paxillin and FAK phosphorylation, HSC-3 shRNA Control and HCS-3 shRNA FSCN cells were starved and cultured for 5, 15 and 30 min with media containing 50 ng/ml of EGF (Sigma-Aldrich, USA).

Anti-FAK, anti-Pyk2, the anti-phosphotyrosine antibody, 4G10, and HRP-conjugated goat anti-mouse and mouse anti-rabbit light chain specific IgGs were all obtained from Millipore (Lake Placid, NJ, USA); normal rabbit and mouse IgGs and RGD peptide were from Santa Cruz (CA, USA), while anti-PLCγ2 and anti-Hic-5 were from Cell Signaling Technology, Inc. (Boston, MA, USA). Anti-Rac1 was from Tebu-Bio (Peterborough, UK). Cross-linked collagen related peptide (CRP) was purchased from Prof. Richard Farndale (Dept of Biochemistry, Cambridge University, UK). The pharmacological inhibitors, PF-573228 (hereafter referred to as PF-228), PP2, Wortmannin, EHT-1864, U73122, GF109302× and the Ca²⁺ chelator, BAPTA, were from Tocris Bioscience (R&D Systems Europe, UK). Tyrphostin A9 was from Calbiochem. ML171 (2-acetylphenothiazine), and BAY61-3606 (hereafter referred to as BAY) were purchased from Sigma Aldrich (St. Louis, MO, USA).

All three FAK family inhibitors (PF-431396, PF-562271, and PF-573228) and Pan-Src kinase inhibitor (Saracatinib) were purchased from Cayman Chemicals. Src kinase inhibitor PP2 and Smad3 inhibitor (SIS3) were purchased from Tocris of R&D Systems. ROCK inhibitor (Y-27632), Cdc42 GTPase inhibitor (ML-141), and Rac1/3 inhibitor (EHop-016) were purchased from Selleckchem. YAP-TEAD inhibitor (Verteporfin) was purchased from Cayman Chemicals (USA) and Selleckchem. Anti-FAK was purchased from Millipore. Antibodies against p-FAK(Y397), p-PYK2(Y402), PYK2, c-Src, p-Src(Y416), Smad2, p-Smad2(S465/467), p-Smad3(S423/425), Smad3, and YAP/TAZ were purchased from Cell Signaling Technology. anti-α-SMA and anti-actin were Sigma-Korea. Antibodies to detect p-PYK2(Y402), p-PYK2(Y579/580), and p-PYK2(Y881) were purchased from Invitrogen. Anti-CTGF and anti-myc-tag were purchased from Abcam. Anti-YAP and anti-α-SMA were purchased from Santa Cruz Biotechnology and Dako, respectively. Secondary antibodies to detect the primary antibodies were purchased as followed; HRP-conjugated anti-rabbit and anti-mouse from Pierce-Thermo Scientific, all Alexa-fluorophore conjugated antibodies from Invitrogen.

Total cell lysate from 2D monolayer culture was prepared by extracting cells with modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with 50 mM NaF, 1 mM sodium pervanadate, and protease inhibitors. Total cell lysate from 3D culture was prepared by incubating cells at 4 °C in the modified RIPA buffer supplemented with 0.5% SDS and protease/phosphatase inhibitor cocktail (Pierce). The extracts were clarified by centrifugation at 13,000 rpm for 30 min and the supernatant was used for immunoblotting. Sources of different antibodies were: anti-Pfn1 (Abcam), anti-phospho-FAK (Y397) (Invitrogen), anti-Smad3 (Biorad), anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Smad3 (S423/S425) (Cell Signalling), anti-Pfn2 and anti-FAK (Santa Cruz), and anti-GAPDH and anti-tubulin (Sigma-Aldrich) Immunoblotting concentrations for different antibodies were: 1:3000 for anti-Pfn1, anti-GAPDH and anti-tubulin; 1:500 for anti-Pfn2, anti-ERK1/2, anti-pERK1/2 and anti-FAK; and 1:1000 for anti-Smad3, anti-pSmad3 and anti-pFAK.