Ffp39

PGCs were collected and lysed using Radio-Immunoprecipitation Assay (RIPA) lysis solution (Beyotime-P0013B, Shanghai, China). Extracted total proteins were separated and transferred to PVDF membrane (Beyotime-FFP39, Shanghai, China) via electrophoresis. Blots were blocked using 5% skimmed milk at room temperature for 2 h, followed by the incubation of primary antibody solution (PI3K (BIOSS-bsm-33219M, Beijing, China), p-PI3K (BIOSS-bs-6417R, Beijing, China), AKT (Affinity-AF6261, Liyang, China), p-AKT (BIOSS-bs-0876R, Beijing, China), GAPDH (Proteintech-10494-1-AP, Wuhan, China)) at 4 °C overnight. After 3 washes with TBST, the blots were incubated with secondary antibody solution (Goat Anti-Mouse IgG (CWBIO-CW0102, Taizhou, China), Goat Anti-Rabbit IgG (CWBIO-CW0103, Taizhou, China) at room temperature for 2 h. The blots were washed 3 times with TBST (Solarbio-T1082, Beijing, China) and subjected to imaging using chemiluminescent substrate.

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).