Western Blot Analysis of RSV-Treated Cells

The control samples consisted with MCF-7 and A549 cells treated with different doses of RSV or DMSO. Total cell proteins were prepared at 24 h post treatment using cell lysis buffer (Cell Signaling Technology, Danvers, USA) supplemented with a cocktail of proteinase inhibitors (Sigma-Adrich). The Western blot analysis was performed as described in a previously published study by our research group [21 (link)]. Briefly, total protein was resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membranes (Millipore, USA) and blocked with 5% nonfat dry milk. The membrane was incubated with a primary antibody, followed by a secondary antibody coupled to HRP. The target bands were developed with ECL substrate (WBKLS0500; Millipore, USA). All experiments were repeated at least three times. The band intensities in the immunoblotting were determined by ImageJ software.