Anti phospho erk thr202 tyr204 antibody

Cells were seeded at a density of 8 × 10³ cells·well⁻¹ of a 96-well plate. Prior to growth factor treatment, the cells were serum-starved for 16 h and then EGF was added. After a certain period, the cells were fixed with 3% paraformaldehyde/PBS for 30 min, permeabilized with 0.5% triton X-100/PBS for 5 min and blocked with 10% FBS/Blocking ONE solution (Nacalai Tesque, Kyoto, Japan) for 1 h. Next, the cells were immunostained using anti-phospho-ERK (Thr202/Tyr204) antibody (#4370; Cell Signaling Technology, Beverly, MA, USA) and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Immunostained images and bright field images were photographed using In Cell Analyzer 2000 (GE Healthcare). Cell areas were automatically determined from bright field images, nuclear areas were determined from DAPI images and, finally, phospho-ERK intensities of each cell and nuclear region were calculated for at least 1200 cells in each condition. These image analyses were carried out using developer toolbox software (GE Healthcare).