Primary antibody against nf κbp65

Cells were fixed with 4% paraformaldehyde (PFA, ThermoFisher, cat. no. 28908) in 1X PBS for 10 min, and then, permeated with 0.1% Triton X-100 (Sigma-Aldrich, cat. no. T8787) in 1X PBS for 15 min and blocked with 5% bovine serum albumin (BSA, PAN-Biotech, cat. no. P06–139310) in 1X PBS-0.1% TWEEN20 (Sigma-Aldrich, cat. no. P2287) solution for 30 min. Samples were incubated with a primary antibody against NF-κBp65 (1:200, Abcam, cat. no. ab1652) overnight, followed by a secondary antibody (1:500, Invitrogen, cat. no. a11036) and Hoechst 33342 (1:1000, Invitrogen, cat. no. H3570) for 1 h in the dark. After three washes with 1X PBS, slides were observed under a Leica DMi8 microscope.

Lung tissues were lysed using RIPA lysis buffer (Beyotime Tech, China). After quantification using a BCA kit (Bio-Rad, USA), an equal amount of proteins was loaded to a 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes using a Trans-Blot Turbo blotting system (Bio-Rad). The membranes were blocked by 5% skim milk and incubated with primary antibodies overnight with the desired dilution concentration. After being washed three times in Tris-buffered saline containing 0.1% Tween 20 (TBS-T, pH 7.5), they were treated with corresponding secondary antibodies. Membranes were then washed three times with TBS-T and the protein bands were visualized using enhanced chemiluminescence (ECL, Thermo Fisher) solution and photographed using a ChemiDoc XRS Plus image analyzer (Bio-Rad). Primary antibody against NF-κB p65 was purchased from Abcam (China) and antibodies against IL-1β, IL-6, TNF-α, VCAM-1, and ICAM-1 were purchased from Cell Signaling Technology (USA). GAPDH was used as a loading control and its specific primary antibody was purchased from Santa Cruz Biotechnology (USA).