Nuclear extract kit

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).