Proteinase and dephosphorylation inhibitors

We measured the concentrations of 8-oxo-29-deoxyguanosine (8-OHdG), a marker of
DNA oxidation in serum using an ELISA kit (Nikken SEIL Corporation, Shizuoka,
Japan) according to the manufacturer’s instructions. The mean values of
duplicate assays with each sample were used for the statistical analyses.
ELISA kits (R&D Systems) were used to detect the contents of transforming
growth factor β (TGF-β), interleukin-1beta (IL-1β), interleukin-6 (IL-6), C-C
Motif Chemokine Ligand 8 (CCL8) in serum and lung tissues according to the
manufacturer’s instructions. Briefly, the lung tissues were homogenized using
Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific)
consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher
Scientific). Then, lung lysates and serum were added to each well and measured
per the manufacturer’s instructions. The optical density of each well was
measured at 450 nm using a microplate reader (Multiskan Fc, Thermo Fisher
Scientific).

Western blot was performed as previously described [25 (link)]. Briefly, lung tissue sample was homogenized using Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total tissue protein purified from lungs were separated by SDS-PAGE gels and then transferred to 0.22-μm PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies against NF-kB p65 (1:500 dilution, Abcam), IκBα (1:1000 dilution, CST), TGF-β (1:1000 dilution, Abcam), pSmad2 (1:1000 dilution, Abcam), α-Tubulin (1:1000 dilution, CST), or GAPDH (1:1000 dilution, Abcam), respectively; and followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako). The expression was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific). Semiquantitative analysis was done using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences).