*2.8. Immunoblot Analysis*

HEp2 cells were seeded in six well cell culture plates (5 <sup>×</sup> 105 cells/well) and incubated for 12 h. Medium was changed to DMEM containing 1% FBS and cells were infected with RSV-GFP (0.1 MOI) for 2 h. Cells were treated with PAE, CTE or acteoside once replacing the medium with DMEM containing

10% FBS. Cells were harvested at 0, 12, 24, 36, 48, hpi and subjected to immunoblot analysis. Briefly, harvested cells were lysed with lysis buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0 and a protease inhibitor (Sigma). Whole cell lysates were mixed with 10x sample buffer (Sigma, St. Louis, MO, USA) at 1:1 ratio, and the total protein was separated in 12% gel by SDS-PAGE and transferred to a PVDF membrane (BioRad, Hercules, CA, USA). The membrane was blocked in 5% bovine serum albumin (BSA, Sigma) and incubated with anti RSV-G antibody or anti-β-actin antibody with 5% BSA and TBST (Tris-buffered saline (LPS Solution) + Tween 20 (Life science, #0777-1L, Suwanee, GA, USA)) respectively. Proteins were detected by incubating with a secondary anti-rabbit IgG-HRP or anti-mouse IgG-HRP for 1 h at room temperature. The membrane was developed with ECL reagent mix (LPS solution, FEMTO-100) and images were captured with an Enhanced Chemiluminescence Detection (ECL) system (GE Life science, Pittsburgh, PA, USA), using Las-4000 mini lumino-image analyzer (GE Life Science). Band intensity was calculated using ImageQuant LAS 4000 control software (Pittsburgh, PA, USA).
