Anti tgfβ1

Anti-NOX-2, anti-Rac-1, anti-p47phox, anti-p-53, anti-Bax, anti-Bcl-2, anti-cytochrome c, anti-β-actin, anti-p-I-κBα, anti-p-p38, anti-p-NF-κB, anti-COX-2, anti-IL-8, anti-TGFβ1, anti-p-ERK, anti-Sp1, anti-CTGF, anti-FGF2, anti-uPA, anti-MMP-2, anti-MMP-9, and anti-α-SMA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA).

The cells were washed twice in PBS and then lysed with protein extracted by IP lysis buffer (Thermo Fisher Scientific). Protein concentration was determined by Pierce detergent compatible Bradford assay kit (Thermo Fisher Scientific). A total of 30 μg protein was loaded for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to a polyvinylidene difluoride membranes (Millipore, Billerica, U.S.A.). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline and then incubated with primary antibodies (anti-Caspase3, anti-Caspase8, anti-Caspase9, anti-cleaved Caspase3, anti-cleaved Caspase8, anti-cleaved Caspase9; Cell Signaling Technology, Shanghai, China and anti-TLR4, anti-IL-6, anti-TGF-β1, anti-pSmad2, anti-Smad2 and anti-GAPDH; Thermo Fisher Scientific) according to the manufacturer’s instructions, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The reactive bands were visualized by an enhanced chemiluminescence substrate solution (Millipore) according to the manufacturer’s instructions.