0.22 μm polyvinylidene fluoride

The hRPR cells were washed three times with ice-cold PBS (4°C, pH 7.4) for 5 min at room temperature and prepared using a protein extraction kit and a protease inhibitor kit (Pierce, Rockford, IL). After centrifugation, the supernatant was collected, and the protein content of each lysate was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce) according to the manufacturer’s instructions. Equal amounts of protein (40 μg) were separated by a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a 0.22-μm polyvinylidene fluoride (PVDF) membrane (Millipore). The primary antibodies used to probe the membranes included anti-UBE3D (1:500, PAB21883, Abnova, USA), anti-CyclinB1 (1:1,000,# 4138, CST, USA), anti-caspase-3 (1:500, ab13847, Abcam, Cambridge, MA, USA), anti-p38 mitogen-activated protein kinases (p38MARK) (1:1,000, #8690, CST, USA), anti-phosphorylated p38 MAPK (p-p38MARK) (1:1,000, #4511, CST, USA), anti-P62 (1:2,000, PM045, MBL, Japan), anti-light chain3 (LC3) (1:1,000, #2775, CST, USA), anti-Beclin1 (1:2,000, ab207612, Abcam, Cambridge, MA, USA) and anti-β-actin (1:2,000, #4970 CST, USA). The membranes were washed and incubated with peroxidase-conjugated secondary antibodies (1:5,000, Boster, China). The proteins were visualized with enhanced chemiluminescence western blot detection reagents (Millipore).

We used lysis buffer, which contained phenylmethylsulfonyl fluoride (Roche), protease inhibitor cocktail (Roche), and the mammalian protein extraction reagent RIPA (Beyotime, China), to extract the total protein from the lung tissue. Then, the total concentration of the extracted protein was measured using the Bio‐Rad protein detection kit (KeyGEN Biotech). A 30‐μg protein sample was subjected to 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to a 0.22‐μm polyvinylidene fluoride (Millipore) membrane, and incubated with specific antibodies (NF‐κB p105: total form, 1:1000, #13586, phosphorylated form, 1:1000, #4806; NF‐κB p65: total form, 1:1000, #8242, phosphorylated form, 1:000, #3033; and IκBα: total form, 1:000, #4814, phosphorylated form, 1:1000, #4806 [Cell Signaling Technology]; GAPDH, 1:10,000, ab181602 [Abcam]). An ECL detection system (Tanon) was used to examine the protein bands after incubating the secondary antibodies.