Ecl western blot analysis system

Total protein was extracted from rAd-FAP-α DCs, rAd-c DCs, LLC cells or CAFs using RIPA buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, 10 mM NaF, 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol] (eBioscience; Thermo Fisher Scientific, Inc.) on ice for 30 min. Protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (20 µg) were separated by 6% SDS-PAGE and transferred onto Hybond-polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in PBS at room temperature for 1 h and subsequently incubated with primary antibodies against FAP-α (1:1,000; cat. no. NB110-85534; Novus Biologicals, Ltd.) and GAPDH (1:1,000; cat. no. NB100-56875; Novus Biologicals, Ltd.) for 2 h at room temperature. Membranes were washed three times in TBST (5 min/wash) and further incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (1:2,500; cat. no. ADI-SAB-301-J; Enzo Life Sciences Inc.) for 1 h at room temperature. Protein bands were visualized using the ECL western blot analysis system (GE Healthcare).

Proteins were extracted from the lung tissue or cells using T-PER Tissue Protein extraction reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Primary antibodies of TLR5, IKKα, p-IKKα, IκBα, p-IκBα, NF-κB, p-NF-κB, IL-1β, TNF-α, IL-18 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology) were used in the study. The immunoactive proteins were detected by using an enhanced chemiluminescence western blot detection kit. The bands were observed using an ECL western blot analysis system (GE Healthcare, Pittsburgh, PA, USA) and exposed to Kodak X-ray film.
Analysis of qPCR was performed as previously described (17 (link)). Fold induction values were calculated using the to 2^−ΔΔCq method, where ΔCq represents the differences in cycle threshold number between the target gene and GAPDH. ΔΔCq represents the relative change in the differences between the control and treatment groups. The primers used in the study are shown in Table I.

Protein expression was determined by western blot analysis. Briefly, the cells were lysed in a lysis buffer (20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% Nonidet P 40, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 mM protease inhibitor cocktail and 10% phosphatase inhibitor cocktail). The proteins present in the cell extracts were quantified using a BCA assay and proteins (50 µg/lane) from each sample were resolved by SDS-PAGE on a 10% gel. This was followed by transference onto a nitrocellulose membrane. The membrane was then treated with non-fat milk (5%) in PBS, and then incubated with a suitable primary antibody: B-cell lymphoma 2-associated X protein (Bax; cat. no. sc-6236) and Bcl-2 (cat no. sc-509) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) overnight at 4°C (dilution 1:1,000), followed by incubation with horseradish peroxidase-conjugated (cat. no. 9003-99-0) and anti-rabbit secondary antibody (cat. no. sc-2372) (dilution 1:1,000) for 1 h at room temperature. The western blots were then observed in an ECL western blot analysis system (GE Healthcare Life Sciences, Chalfont, UK).