Western Blotting of Erk Phosphorylation

Oct4-GFP mESCs were used for all stimulation experiments. Cells were plated (1 × 10⁵ cells/cm²) onto gelatinized six-well plates in mESC growth medium. After 8–12 hours, cells were washed with DPBS, and serum starved (~18 hours) by replacing with FBS-free mESC growth medium. Stimulation was performed by adding FGF2 (25 ng/ml) in FBS-free mESC media to cell monolayers for 10–15 minutes at 37°C, 5% CO₂. The plate was then immediately incubated on ice, and total protein was extracted using 1× cell lysis buffer and scraping. After 10 minutes of incubation on ice, the mixture was centrifuged at 16,000g for 10 minutes (4°C) to pellet and remove insoluble components. The supernatant was subjected to a biscinchoninic acid (BCA) assay to quantify total protein levels, and upon normalization, 10 μg of protein was separated by SDS-PAGE (10% Tris-Glycine-sodium dodecyl sulfate- polyacryl-amide gel electrophoresis) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Anti-phosphoErk and anti-Erk antibodies were used to probe for levels of phosphorylated and total Erk protein levels. Densitometry was performed using ImageJ.