Wl01506

The cells were lysed with Ripa buffer (wanleibio, China) containing 10 mM protease inhibitor (PMSF; wanleibio, China). The lysate was centrifuged at 12,000 rpm and 4 °C for 10 min. The precipitation was separated and the protein concentration was determined by the BCA protein concentration determination kit (wanleibio, China). The equivalent protein (40 μg) was added to the 8–15% SDS-PAGE gel and then transferred to the PVDF membrane (Millipore, Billerica, USA). The membrane was sealed in 5% skimmed milk powder solution for 1 h, and then incubated overnight at 4 °C with antibodies specific for LC3 (1:500; wl01506; wanleibio, China), runx2 (1:500; wl03358; wanleibio, China), collagen I (1:500; wl0088; wanleibio, China), β-actin (1:1000; wl01845; wanleibio, China). After washing 4 times with TBST, the membrane was incubated with goat anti-rabbit IgG-HRP (1:5000; WLA023; wanleibio, China) for 45 min at room temperature. The protein bands were visualized using ECL detection kit (wanleibio, China) and compared on the same membrane. We used gel image processing system (Gel-Pro-Analyzer software) to analyze the optical density of target strips and calculate the proportion of LC3-II/I in order to determine the level of autophagy.

Cells were lysed in RIPA lysis buffer (catalog no. P0013B, Beyotime) coupled with PMSF (catalog no. ST506, Beyotime) at a final concentration of 1 mM, and the protein concentration was determined using a BCA Protein Assay Kit (catalog no. 23227, Thermo). Protein (20 μg) from each sample was separated by 10% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (catalog no. IPVH00010, Millipore). Five percent of skim milk (catalog no. 232100, BD) was used to block the nonspecific binding for 1–2 h at 37 °C and then incubated with anti-LC3I/II (1:1000, catalog no. WL01506, Wanleibio), anti-p62 (1:1000, catalog no. 5114S, CST), anti-p65 (1:1000, catalog no. 1074S, CST), anti-P mTOR (1:1000, catalog no. 5536S, CST), anti-mTOR (1:1000, catalog no. 20657-1-AP, Proteintech), anti-β actin (1:1000, catalog no. ab8226, abcam) primary antibody at 4 °C overnight. The membrane was washed with PBST buffer and then incubated with HRP secondary antibodies (1:10000, catalog no.111-035-045, Jackson) for 2 h at 37 °C. After washing with PBST buffer six times, the membrane was exposed with enhanced chemiluminescence (ECL) reagents (catalog no. SB-WB012, Share-bio) and the Millipore ECL system, and then gray scanning was performed using TanonImage (4600, Tanon).

Primary anti-VEGFA (ab52917, Abcam, USA), anti-vWF (PB0273, Boster, Wuhan, China), anti-PCNA (BM0104, Boster, Wuhan, China), anti-LC3 (WL01506, Wanlei, Shenyang, China), anti-p62 (WL02385, Wanlei, Shenyang, China), anti-ERα (ab32063, Abcam, USA), and anti-PR (sc-810, Santa Cruz, TX, USA) antibodies were diluted in 0.5% goat serum in PBS. The sections were heated in a microwave in sodium citrate solution for antigen recovery and pretreated with 0.3% H₂O₂ in methanol to quench endogenous peroxidase activity. Then, the samples were incubated with 3% goat serum to block nonspecific antibody binding sites. Subsequently, the samples were incubated with primary antibodies at 4 °C overnight. Immunoreactivity was visualized using a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab64264, Abcam) according to the manufacturer’s instructions.