Mouse anti β actin antibody

The protein content in the cellular pellet was separated on SDS-PAGE gels after cellular lysis and boiling for 10 ​min. The separated proteins were transferred onto an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, USA), followed by incubation in PBS containing 5% dried skim milk and 0.05% Tween-20 for 2 ​h at room temperature (RT). Membranes were incubated with the rabbit anti-NS1 and anti-VP6 antibody (prepared by our laboratory, 1:1000 dilution), mouse anti-HA-tag antibody (Proteintech, USA, 1:5000 dilution), and mouse anti-β-actin antibody (Bioworld, USA, 1:5000 dilution), respectively, for detection the expression of BTV NS1 and VP6 protein, oISG20, and β-actin. In the Co-IP assay, the expression of BTV VP1∼VP7, NS1∼NS3 was tested by the mouse anti-Myc-tag antibody (Cell Signaling Technology, USA, 1:1000 dilution). After overnight incubation at 4 ​°C, the membranes were washed with blocking buffer three times, and the membranes were incubated in HRP-conjugated goat-anti-mouse or goat-anti-rabbit secondary antibody (Abcam, USA, 1:10,000 dilution) for 1 ​h at RT. After membranes were washed, the bands were detected by using SuperSignal West Femto kit (Thermo, USA) in a ChemiDoc MP imaging system (Bio-Rad, USA). The band intensity was quantified by the Image J software.