Western Blotting Analysis of Protein Samples

Cells seeded in 6-well plates were harvested after two washes with Phosphate buffered saline (PBS) containing phosphatase and protease inhibitors (Thermo Fischer Scientific, Waltham, MA, USA) and lyzed with RIPA lysis Buffer (Sigma-Aldrich, St. Louis, MS, USA). Proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes [14 (link)]. The membranes were first blocked with 5% milk in TBS-Tween for 1 h, then incubated with appropriate dilutions of primary antibody at 1:1000 for 2 h. Anti-rabbit immunoglobulin-horseradish peroxidase and anti-mouse immunoglobulin-horseradish peroxidase conjugates were used as secondary antibodies (dilution 1:2000, Vectors). The membranes were incubated with Amersham ECL Select or prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) and exposed to a film or on an Amersham imager 600 (GE Healthcare) or GeneGnome imager (Syngene, Cambridge, UK). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Vector Labs. The mouse anti-pan flavivirus envelope E protein mAb 4G2 was produced by RD Biotech. Mouse antibody against HO-1was from Abcam and the mouse antibody against α-tubulin, β-tubulin and M2 FLAG were from Sigma–Aldrich. All Western-blot data are representative of three independent experiments.