Chemiluminescence substrate system

Lung tissues or fibroblasts were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China), to which two kinds of protease inhibitor cocktails (MCE, HY-K0022) had also been added. The proteins were separated on polyacrylamide gels and finally detected with a chemiluminescence substrate system (Bio–Rad Laboratories, CA, United States and Tanon, Shanghai, China) (Miao et al., 2020 (link)). The antibodies used for western blotting were as follows (Table 1).

The proteins from lung tissues have been extracted utilizing the RIPA Lysis Buffer containing protease inhibitor (GRF101, Epizyme) and phosphatase inhibitor cocktails (GRF102, Epizyme) while quantification performed utilizing a BCA Protein Assay Kit (ZJ101, Epizyme). Equivalent amounts of proteins have been electrophoresed on polyacrylamide gels and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated for 2 h at room temperature by the primary antibodies which included ß-Tubulin antibody (HX 1829, huaxingbio), anti-FIZZ1 antibody (1:1,000, ab39626, Abcam) p38 mitogen-activated protein kinase (MAPK) Rabbit mAb (#8690, Cell Signaling Technology), Phospho-p38 MAPK (Thr180/Tyr182) Rabbit mAb (#4511, Cell Signaling Technology). Following a 40-min incubation at room temperature with secondary antibodies, the membranes were detected using a chemiluminescence substrate system (Bio-Rad Laboratories, CA, United States). The band intensities of proteins have been quantitated using Image J software.