Phenylmethanesulfonyl fluoride

Total proteins in each group were extracted using lysis buffer with 1 mM phenylmethanesulfonyl fluoride (Beyotime) on ice and centrifuged at 10000×g at 4 °C for 5 min. Nuclear proteins and plasma proteins were extracted with a nucleoprotein extraction kit (Beyotime). The concentration of proteins was measured with a BCA protein assay kit (Beyotime). Equal amount of proteins from each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blockade, the membranes were incubated with primary antibodies: LC3 (1: 1000), beclin-1 (1: 200), ATG5 (1: 1000), p62 (1: 1000), JAK2 (1: 1000), p-JAK2 (1: 1000), STAT3 (1: 500), p-STAT3 (1: 1000), β-actin (1: 1000) and Histone H3 (1: 2000) overnight at 4 °C. After washing with Tris buffered saline with Tween, the membranes were incubated with corresponding secondary antibodies (1: 5000). The blots were visualized with an enhanced chemiluminescence substrate luminescence kit (Beyotime). Images were captured and analyzed with a Gel-Pro-Analyzer software.

Samples collected from tissues or cells were lysed with cold cell lysis buffer containing phenylmeth-anesulfonylfluoride (Beyotime Institute of Biotechnology) and subjected to SDS-PAGE. The proteins were then electrophoretically transferred onto PVDF membranes, blocked, and then exposed to primary antibodies against PCNA (1:1,000), cyclin D1 (1:1,000) or CDK4 (1:1,000) overnight at 4°C. β-actin (1:1,000) was also measured as an internal control for protein loading. Membranes were then incubated with secondary HRP-conjugated antibodies at 1:2,500 dilution for 2 h at room temperature followed by enhanced chemiluminescence detection.

The SWFs from D580 and D280 hens were homogenized using 500 μL ice-cold RIPA (P1003B, Beyotime, Jiangsu, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China). The cultured GCs were digested with EDTA-trypsin for 2 min and homogenized by 50 μL RIPA containing 1 mM phenylmethanesulfonyl fluoride.

Total protein was obtained from cells and tissues by using RIPA buffer (Beyotime) and phenylmethanesulfonyl fluoride (Beyotime). Nuclear protein was extracted from cells by using a Nuclear Protein Extraction kit (Beyotime) according to the manufacturer’s protocol. Cell lysates (15–30 μg protein) were loaded per lane and separated on SDS-PAGE gels (8%, 10%, or 15% separation gel). Next, the proteins were blotted onto the PVDF membrane. After being treated with 5% bovine serum albumin for 1 h, the membrane was incubated with primary antibodies (Shown in Supplementary Table 2) overnight at 4 °C followed by the incubation of the secondary antibody for 40 min. The secondary antibodies used were as follows: goat anti-rabbit IgG (No. SA00001-2; Proteintech; 1:10000) and goat anti-mouse IgG (No. SA00001-1; Proteintech; 1:10000). After washing with TBST, immunoreactions were visualized by using an ECL reagent solution (7 Sea biotech, Shanghai, China). Quantitative normalization was performed using the expression of β-actin. Histone H3 served as an internal control for nuclear protein detection.

Lungs were excised, immediately frozen at −80°C, and ground in liquid N₂. Cold RIPA extraction buffer (Beyotime, Haimen, China) was added to the pulverized tissue and then the mixture was sonicated. Next, 1 mM phenylmethanesulfonyl fluoride (Beyotime), 2 mM ethylenediaminetetraacetic acid, 10 mM dithiothreitol, and protease inhibitor cocktails (Roche, Basel, Switzerland) were added, after which the mixture was centrifuged at 4°C and 30,000 × g for 15 min. The supernatant was collected and added to five volumes of cold acetone containing 10% (v/v) trichloroacetic acid, thoroughly mixed, and incubated at −20°C overnight. The mixture was centrifuged again at 4°C and 30,000 × g and the supernatant was discarded. The precipitate was then washed three times with chilled acetone, dissolved in RIPA buffer, and air-dried. Proteins were quantified with a BCA kit (Thermo Fisher Scientific, Waltham, MA, United States), after which 300 μg of total protein was mixed with sequencing-grade trypsin (Promega, Madison, WI, United States) at an enzyme-to-protein ratio of 1:50 and incubated at 37°C for 16 h. Peptides obtained from the digestion were dried by vacuum centrifugation.