Western Blot Analysis of MAPK Signaling

For Western blot analyses, cells were plated on 6-well plates and were serum-starved overnight. Cells were treated with compounds accordingly, and protein samples were prepared as described previously [36 (link)]. Then, 30 µg of total proteins was loaded to each well, and proteins were separated using 4–12% Tris Glycine Precast Gel (KOMA BIOTECH, Seoul, Republic of Korea). Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Blocking was carried out using Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% BSA at room temperature for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4 °C with the indicated antibodies: anti-p42/44 (Cell Signaling; Cat#9102S) and anti-phospho-p42/44 (Cell Signaling; Cat#9101L). Subsequently, the membranes were washed out with TBST 3 times at 5 min intervals and incubated with HRP-conjugated anti-secondaries for 1 h at room temperature. Finally, visualization was carried out using an ECL Plus immunoblotting detection system (GE Healthcare, Piscataway, NJ, USA). All experiments were repeated five times independently, and ImageJ software (NIH, Bethesda, MD, USA) was used for analysis.