Ecl high signal reagent

The lower right lung lobes were removed and stored in liquid nitrogen for protein analysis. Briefly, the lung tissues were lysed on ice in a RIPA (P0013B, Beyotime) buffer containing 1 mM PMSF (Beyotime). After sonication, the protein concentration in the supernatants was measured using a bicinchoninic acid protein assay kit (Beyotime). Equal amounts of protein were loaded in 10% SDS–PAGE, and then the proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% bovine serum albumin for 1 h at room temperature. After rinsing in PBST 5 times, the membranes were incubated with each primary antibody overnight at 4°C: the anti-claudin 5 (Abcam, ab15106), the phosphor-MAPK family Antibody sampler kit (cell-signaling technology, #9910) the MAPK family Antibody sampler kit (cell-signaling technology, #9926), and β-actin (cell-signaling technology, #4970). After washing with PBST, the membranes were finally incubated with the appropriate secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL High-Signal reagent (Thermo). The band intensity was quantified using the ImageJ software.

The cells were lysed on ice in RIPA (P0013B, Beyotime) buffer containing 1 mM PMSF (Beyotime). Equal amounts of protein were separated by 10% SDS–PAGE, and then the proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% bovine serum albumin for 1 h at room temperature. After rinsing in PBST five times, the membranes were incubated with each primary antibody overnight at 4 °C, including HMGB1 (10829-1-AP, Proteintech, 1:1000), citrullinated histone H3 (ab5103, Abcam, 1:1000), fibronectin (15613-1-AP, Proteintech, 1:1000), α- SMA (ab5694, Abcam, 1:200), NF-κB p65 (8242, CST, 1:1000), phospho-NF-κB p65 (3033, CS, 1:1000), Smad3 (9513, CST, 1:1000), phospho-Smad3 (9520, CST, 1:1000), elastase (ab68672, Abcam, 1:1000), and β-actin (4970, CST, 1:1000). After washing with PBST, the membranes were finally incubated with the appropriate secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL high-signal reagent (Thermofisher). We confirmed all blots derive from the same experiment and were processed in parallel.