GPER Signaling Pathway Activation

Cells were stimulated with E₂, G1, or TAM for 15 min with or without G15, AG, U0126, and WM pretreatment. Western blotting was then performed as described previously (Liu et al. 2010 (link)). Briefly, cell lysates were harvested in a cell lysis buffer (Boster, Wuhan, China), dissolved in 9% SDS–PAGE buffer, and subjected to western blotting using primary detection antibodies against total or phosphorylated ERK1/2 (diluted 1:1000; BioWorld, St Louis Park, MN, USA) and GPER. Membranes were incubated overnight at 4 °C before incubation with the appropriate HRP-conjugated secondary antibodies. Immunodetection was conducted using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). Optimal density was analyzed using Image J Software, and results were expressed as fold change relative to the control.