2.16. Western Blot

Two of the most downregulated proteins in Flavivirus infections, as determined in the SILAC experiment, were selected for validation by Western blot: HMGA1 and MRPS27. In total, 1 μg (HMGA1) and 10 μg (MRPS27) of total protein extract were separated by one-dimensional SDS-PAGE at 180 V for approximately 1 h before being transferred to a PVDF blotting membrane (GE 10600023) at 100 V for 1 h 15 min. After blocking in 2.5% non-fat milk in PBS for 1 h, the membranes were incubated with a 1:3000 dilution of primary antibody (anti-HMGA1: Abcam ab129153; anti-MRPS27: Abcam ab153940) for 6 h at room temperature, washed three times in TBS-T for 5 min, incubated with a 1:6000 dilution of anti-rabbit HRP secondary antibody (Abcam, Cambridge, UK, ab205718), washed twice in TBS-T and once in PBS for 5 min. Finally, ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA, 1705061) was used to visualize the target proteins. The membranes were stripped by boiling in PBS for 1 min, blocked again, and incubated with anti-β-actin primary antibody (Sigma-Aldrich, St. Louis, MO, USA, A5441) for 1 h at RT followed by an incubation with anti-mouse HRP secondary antibody (NEB 7076) for 1 h at RT.