Quantitative Western Blot Analysis of ELK1

Samples for Western blots were collected, prepared, and analyzed as described previously [31 (link),32 (link)]. Briefly, 661W cells were harvested and lysed in a Tris lysis buffer (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, and 1 Na₃VO₄, pH 7.5. Samples were separated on 10% sodium dodecyl sulfatepolyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 3% BSA in tris buffered saline Tween 20 (TBST) at room temperature for 1 h and incubated in primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated in anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibody (1:1000, #7074S, Cell Signaling, Beverly, MA, USA) at room temperature for 1 h. The blots were visualized using Super Signal West Pico/Femto chemiluminescent substrate (#34078/#34096, ThemoFisher). Band intensities were quantified using Image J (National Institutes of Health; NIH, Bethesda, MA, USA). The primary antibodies used in this study were anti-ELK1 (1:500, #9182S, Cell Signaling) and anti-β-actin (1:2000, #8457L, Cell Signaling). The band intensities of ELK1 were normalized to those of β-actin.