Nf kappab

Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS) and 0.25% Trypsin–EDTA solution were purchased from Gibco-BRL (Grand Island, NY, USA). Tryptase was purchased from Sigma-Aldrich (St. Louis, MO, USA), and it is the human lung Tryptase, which is a neutral serine protease and the predominant protein in mast cell granules. PAR-2 inhibitor FSLLRY-NH2 (FS) was synthesised by CL Bio-Scientific Inc. (Xi An, China). CCK-8, RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). Rabbit anti-PAR-2 polyclonal antibody and fluoroshield mounting medium with 4′,6-diami-dino-2-phenylindole (DAPI) were purchased from Abcam (Hongkong, China). Anti-TLR4 monoclonal antibody, anti-VCAM-1 antibody (EPR5 047) and anti-occludin antibody (EPR8208) were purchased from Abcam (Hongkong, China). Anti-GAPDH antibody was purchased from Bioworld Technology, Inc. (USA). Anti-p44/42 MAPK monoclonal antibody (extracellular regulated protein kinases, ERK), anti-Phospho-p44/42 monoclonal antibody (phosphoERK) and NF-kappa B were purchased from Cell Signaling (Beverly, MA, USA).Anti-rabbit and anti-mouse secondary antibodies were all purchased from Jackson Immuno Research Laboratories Inc. (Boston, MA, USA).

Cells were lysed using radioimmunoprecipitation assay buffer (RIPA: 1 % IgepalCA630, 0.5 % sodium deoxycholate, 0.1 % SDS, 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Sigma Aldrich, Steinheim, Germany). 50 µg of total protein was separated on 4–12 % Nupage bis–tris gels (Life Technologies, Darmstadt, Germany) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The blots were blocked using either 5 % (w/v) BSA or 5 % (w/v) milk in TBST for 1 h and then probed using primary antibodies against the p65 subunit of Nuclear Factor kappaB (NF-kappaB), Inhibitory Protein I-kappa-B-alpha, Caspase-3, PARP or GAPDH (1:1000, Cell Signaling, Danvers, MA) as well as HRP-coupled secondary antibodies directed against rabbit or mouse IgG, respectively (1:2000; Cell Signaling, Danvers, MA). Detection was performed as previously described [20 (link)]. Cytoplasmic and nuclear proteins were extracted using the NE-PER nuclear and cytoplasmic extraction kit (Thermo-Fischer Scientific, Schwerte, Germany).

Anti-TM4SF4 antibody for western blot analysis was purchased from Sigma-Aldrich. Antibodies against phospho-Ser473 AKT, AKT, phospho-ERK, ERK, phospho-NF-kappaB (p65), NF-kappaB, phospho-PI3K, PI3K, PTEN, phospho-Tyr1068 EGFR, EGFR, phospho-IGF1R beta (Tyr1131)/insulin receptor beta (Tyr1146), IGF-1R beta (111A9), and anti-beta-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MMP-2, anti-MMP-7, and anti-MMP-9 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as described previously [37 (link)]. Antibodies against N-cadherin and E-cadherin were purchased from BD Biosciences.