Ecl western blot kit

Nucleoprotein and total protein were extracted from lung tissue or cells using RIPA reagent (Beijing Solarbio Science & Technology Co., Ltd.). A BCA protein assay kit (Thermo Fisher Scientific, Inc.) was used to determine the protein concentration of samples from lysed lung tissues or cells. Each well of a 10% SDS-PAGE gel was loaded with an equal volume of protein, and the proteins were separated by SDS-PAGE, after which they were transferred to a PVDF membrane. After incubation with 5% milk at room temperature for 1 h, the membrane was incubated with primary antibodies (all 1:1,000; all Cell Signaling Technology, Inc.), including anti-NF-κB p65 (cat. no. 8242), anti-p-NF-κB p65 at Ser536 (cat. no. 3033), anti-β-actin (cat. no. 3700) and anti-Lamin B (cat. no. 12255), at 4°C overnight. Then, the membrane was incubated with corresponding secondary antibodies (1:2,000; cat. no. 6990; Cell Signaling Technology, Inc.) at room temperature for 1 h. The protein bands were visualized using an ECL western blot kit (Bio-Rad Laboratories, Inc.) and visualized on a ChemiDoc XRS System (Bio-Rad Laboratories, Inc.). Protein expression was calculated by measuring the optical density using Image Lab software (version 3.0, Bio-Rad Laboratories, Inc.). Target protein expression levels were normalized to those of β-actin.