Pmsf buffer

The effective concentrations of siULL1 and JNK inhibitor were selected for exploring the role of UFL1 in the process of milk protein and fat synthesis. siUFL1 was transfected by Lipofectamine 2000 reagent. The concentration of 20 μM of SP600125 used in the starvation medium for 2 h suppressed the JNK expression. After SP600125 treatment, proteins from the treated samples were collected and analyzed by western blotting. Cells were lysed in 200 μL lysis buffer containing RIPA buffer (P0013B; Beyotime Biotechnology) and PMSF buffer (ST506, Beyotime Biotechnology). Protein concentrations were determined using the BSA assay kit (P0010; Beyotime Biotechnology). Equal amounts of protein samples (30–60 μg) were separated using 12% SDS-PAGE and electrotransferred onto PVDF membranes (FFP39, Beyotime Biotechnology). Membranes were blocked with 5% free-fat milk for 2 h at room temperature and subsequently incubated with primary antibodies at 4°C overnight (Table 1). After three washes with PBS, membranes were subsequently rinsed with TBST for 5 min and incubated with secondary antibodies for 2 h at room temperature. The membranes were visualized using LAS-4000 (Fujifilm, Tokyo, Japan).

Spinal cord tissues (1.5 cm length from the injury epicenter) and cells were collected for protein assay. The tissues and cells were homogenized in RIPA lysis buffer containing PMSF buffer (P0013B, Beyotime, Beijing, China) for 30 min on ice. After centrifugation at 12,000 RMP (25 min, 4°C) to remove debris, the supernatant was quickly stored at −80°C. Extracts were first quantified with bicinchoninic acid Protein Assay Kit (P0010, Beyotime, Beijing, China). Then, tissue samples containing 40 μg of protein were separated by sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the appropriate primary antibodies overnight, after which they were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Finally, bands were detected by BeyoECL Plus (Beyotime, Beijing, China), and signals visualized by a Tanon 5500 Gel Imaging System (Tanon, Shanghai, China).