P nfκb p65 ser 276

Eca109 (esophageal cancer), MCF7 (breast cancer) and normal human lung fibroblasts (NHLF) cells were cultured according to ATCC. JQ1 was purchased from Selleckchem and formulated in DMSO. The following primary antibodies were used: BRD4 (WB:1:1000 dilution, Abcam), c-MYC (WB: 1:200 dilution, Santa Cruz), Collagen 1a (Col1) (WB: 1:1000 dilution, Abcam), TGF-β (WB: 2 μg/ml, Biorbyt), p-NFκB p65 (ser 276; WB: 1:500 dilution, Santa Cruz), NFκB p65 (WB: 1:500 dilution, SantaCruz), p-Smad2 (Ser465/467) (WB: 1:1000 dilution, CST), p-Smad3 (Ser423/425) (WB: 1:1000 dilution, CST), Smad2/3 (WB: 1:500 dilution, Santa Cruz), and GAPDH (WB: 1:3000 dilution, Abcam), α-smooth muscle actin (α-SMA) (IF: 1:500; Abcam).

Lung protein extracts were prepared using the Nuclear Extract Kit (BestBio, China) following the manufacturer's instruction. Equal amounts of protein were separated by SDS-PAGE and subsequently blotted on polyvinylidene fluoride membranes (220 mA, 65 min). Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C. The following antibodies and dilutions were used: TLR7 (Catalog number 2633, Cell Signaling Technology; 1 : 1000); MyD88 (D80F5) (Catalog number 4283, Cell Signaling Technology; 1 : 1000); TRAF6 (Catalog number 04-451, Millipore, USA; 1 : 1000); IκB-α (c-21) (sc-371, Santa Cruz Biotechnology, 1 : 200); p-IKKα/β (Ser 176) (sc-21661, Santa Cruz Biotechnology, 1 : 200); NF-κB p65 (sc-109, Santa Cruz Biotechnology, 1 : 200); p-NF-κB p65 (Ser 276) (sc-101749, Santa Cruz Biotechnology, 1 : 200); and GAPDH (14C10) (Catalog number 2118, Cell Signaling Technology; 1 : 1000). The membranes were blotted with appropriate secondary antibodies (Immunology Consultants Laboratory, Inc., USA; 1 : 5000), and the blotted proteins were visualized by enhanced chemiluminescence using a commercially available kit (Millipore, USA).