4.8. Western blotting analysis

AMPK and ACC antibody sampler kits were purchased from Cell Signalling (Massachusetts, USA). Anti‐GAPDH antibody from Sigma (Shanghai, China) and anti‐D2R, anti‐ERα and anti‐ERβ antibodies were purchased from Santa Cruz (Texas, USA). For cell experiments, 1 × 10⁷ cells were harvested, washed twice with ice‐cold PBS, lysed in RIPA lysis buffer with 100 mmol/L phenylmethanesulfonyl fluoride, and then centrifuged (12 000 rpm; 4°C; 20 minute) to collect the supernatants.

Samples of xenograft tumours and human PRLomas, were homogenised on ice in RIPA lysis buffer containing 100 mmol/L phenylmethanesulfonyl fluoride. Supernatants were collected after centrifugation.

We separated 10 μg of extracted protein on 12% SDS‐PAGE gel, which was transferred onto an Immobilon‐P membrane. Membranes were blocked in Tris‐buffered saline with 5% non‐fat milk, then probed with designated primary antibodies overnight at 4 °C and incubated with the relevant peroxidase‐conjugated secondary antibody (1:5000) for 1 hour at room temperature. Next, membranes were washed and visualised with an enhanced chemiluminescence system. The AMPK, D2R, ERα, and ERβ expression levels were quantified and normalised to the GAPDH control.