Evaluation of Protein Regulation in RF-EMF Exposed Cells

After exposure to RF-EMF, cells were harvested, and protein lysates were prepared as previously described [20 (link)]. Histones were extracted with 0.2 M HCl and neutralized with 1 M NaOH. Lysates or histones with 15–20 μg of protein were separated on 10–15% SDS polyacrylamide gels (PAGE) and electro-transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with primary antibodies including anti-phospho-Erk1/2, anti-Erk1/2, anti- β-actin, anti-histone3 (Cell signaling Technology, Inc.), and anti-phospho-H2AX (Millipore, Germany). After primary antibody incubation, membranes were then treated with the appropriate secondary antibodies for 1 h at room temperature. Protein bands were visualized by chemiluminescence detected with AmershamTM ImageQuant 800TM (Cytiva).