Western blot analysis of the phosphorylation of ERK1/2

HEK293 and MCF-7 cells transiently transfected with kisspeptin receptor were used for phosphorylation assay of the extracellular signal-regulated kinase 1/2 (ERK1/2) After 24 hours following transfection, the cells were serum-starved in a serum-free medium (DMEM supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1% (w/v) bovine serum albumin (BSA), 2 nM L-glutamine, 0.2 unit/ml penicillin and 0.2 μg/ml streptomycin) overnight prior to any treatment. Assays were then conducted by stimulating the cells with KP-10 or KP phosphinic peptides. Following stimulation, the cells were placed on ice and solubilized with SDS-PAGE sample loading buffer supplemented with 5 mM sodium orthovanadate and complete^TM protease inhibitor cocktail. Solubilized samples were boiled and then subjected to SDS-PAGE. Subsequently, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes for immunoblotting. Immunoblots were then assayed for phosphorylated ERK1/2 using a phospho-specific rabbit monoclonal antibody (1:1000) (phospho-p42/44 ERKs, Thr²⁰²/Tyr²⁰⁴) and for β-actin using a mouse monoclonal antibody (1:1000). Following incubation with IRDye® goat anti-mouse (1:5000) or anti-rabbit (1:10000) IgG (H + L) secondary antibody, the PVDF membranes were visualised using Odyssey® infrared imaging system (LI-COR, Lincoln, USA). Immunoblots were quantified by densitomery using the Odyssey® application software (version 3.0).