Protein extraction solution

Naïve CD4⁺ T cells were lysed in protein extraction solution (Solarbio) plus protease and phosphatase inhibitor (Solarbio). Proteins were quantified using BCA reagent (Servicebio). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out using precast gel (Willget Biotech), and proteins were transferred onto polyvinylidene fluoride membranes (Servicebio). The membranes were incubated with 5% BSA (37°C, 1.5 h), then incubated with PCSK9 (1:500), p‐ERK (1:1500), ERK (1:3000), p‐JNK (1:1500), JNK (1:3000), p‐NF‐κB p65 (1:1500), NF‐κB p65 (1:3000), or GAPDH antibody (1:8000) (All bought from Abcam; 37°C, 1 h). Afterward, membranes were incubated with secondary antibody (1:5000; 37°C, 1 h; Servicebio). The ECL Kit (Servicebio) was used for detection of blots.

Adipocytes were harvested in protein extraction solution (Solarbio) and incubated for 30 min at 4°C. After cell debris was removed, supernatant containing proteins were collected and determined by using the Pierce BCA protein assay kit according to manufacturer instructions. Next, 40 μg proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. Blots were incubated by using 5% milk blocking solution at room temperature overnight with a primary antibody against AMPK and p-AMPK. The blots were then washed with TBST and incubated with the horseradish peroxidase-conjugated secondary antibody (1:5000) for an hour. Finally, the blots were developed using an enhanced chemiluminescence kit (Salarbio) according to manufacturer protocols.

Testosterone propionate (TP) was purchased from Ningbo Second Hormone Factory (Ningbo, Zhejiang, China), and FN was purchased from Zhejiang CONBA Pharmaceutical Co., Ltd. (Hangzhou, Zhejiang, China). LY was purchased from Xinjiang Keyu Technology Co., Ltd. (Urumchi, Xinjiang, China). CUR was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). BPH-1 human benign prostatic hyperplasia cell line cells were purchased from Beina Biology (Beijing, China). ELISA kits for determining rat dihydrotestosterone (DHT), 5α-reductase, testosterone (T), estradiol (E2), prostate-specific antigen (PSA), interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were purchased from Jiangsu Meibiao Biotechnology Co., Ltd. (Yancheng, Jiangsu, China). Protein extraction solution was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).