Ripa reagent

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.

The protein was extracted using RIPA reagent (Solarbio, Beijing, China) with 1 mM PMSF (Solarbio), quantified via a BCA assay kit (Amyjet, Wuhan, China) and denatured at 100°C for 10 min. Next, 20 μg protein samples were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Solarbio). After the transfer, the membranes were interacted in 5% non-fat milk and incubated with specific primary antibodies (Abcam) as well as secondary antibody. The antibodies used in this research were listed as: anti-CDK4 (ab137675, 1:2000 dilution), anti-Cyclin D1 (ab226977, 1:3000 dilution), anti-Snail (ab216347, 1:500 dilution), anti-Vimentin (ab137321, 1:2000 dilution), anti-E-cadherin (ab15148, 1:500 dilution), anti-HK2 (ab227198, 1:5000 dilution), anti-CHD1 (ab242098, 1:1000 dilution), and anti-β-actin (ab227387, 1:5000 dilution), and horseradish peroxidase-conjugated IgG (ab97051, 1:5000 dilution). The protein bands were visualized via ECL reagent (Abcam). The relative protein expression was normalized to β-actin level and the control group.

Total protein from the COV434 cells and mouse ovaries was extracted using RIPA reagent (Solarbio, China) supplemented with protease inhibitor cocktails (Abmole, China). After being denatured and separated on a 10% polyacrylamide SDS-PAGE gel, proteins were transferred to a Polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Protein expression was analyzed with indicated primary antibodies by using goat anti-rabbit or anti-mouse HRP-conjugated secondary antibodies. The protein bands were visualized using a electrochemiluminescence (ECL) reagent kit (EpiZyme, China) and imaged with Bio-Rad Gel Imaging Systems (Bio-Rad, USA). Antibodies used are indicated in Supplementary Table 2.