Ceb lysis buffer

Human adenocarcinoma cell lines A549, H358 and human immortalized small airway epithelial cell line (HPLD-1) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA). The cell lines were routinely subcultured every 3–5 days. All siRNA transfections were performed using Dharmafect1 # T-2001-03 (Thermo Fisher Scientific Inc, Pittsburgh, PA, USA) as per manufacturer's protocol. After total 72 hrs of transfection cells were harvested in CEB lysis buffer # FNN0011 (Invitrogen, Life technologies, Grand Island, NY, 14072). Protein was quantitated by using Pierce's BCA Protein Assay Reagent Kit (# 23227) from Pierce Biotechnology, Rockford, IL, USA as per manufacturer's protocol (for more detail regarding siRNA sequences used for study, see Supplemental Methods).

For experimental procedures, cells in the log phase of growth were harvested and washed twice with ice-cold PBS and harvested in CEB lysis buffer (Invitrogen, Life Technologies, and Grand Island, NY, 14072). Protein was quantitated using Pierce BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford IL, USA) as per manufacturer's protocol. After protein extraction from cells and tumor tissues (9g), an equal amount (20 μg/well) of proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose (NC) membrane guided by a pre-stained protein molecular weight ladder in a wet transfer system (Beijing Liuyi Biotechnology, China). The membranes were then blocked with 5% fat-free milk in TBST at room temperature for 1 h and probed with specific primary antibodies against PTX3, Sox2, Oct4, N-cadherin, Snai1, E-cadherin, Nav 1.5, NF-κB, p65/NF-κB, p-p65, IκBα, IKKα, TNF-α, β-catenin, GSK3-β, pGSK3-β, PRAP, and GAPDH at 4°C overnight. This was followed by incubation with appropriate species-specific secondary antibodies at room temperature for 1 h. Antibody binding was detected with an enhanced chemiluminescence kit (ECL, Amersham, UK) and detected in a ChemoDocTM XRS + Imager system (Bio-Rad, USA). Relative quantities were analyzed with Image Lab software v4.0.1 (Bio-Rad, USA).