Rabbit anti stat1

The assay was performed as described previously (Reid et al., 2006 (link)). Briefly, Vero cells were seeded onto 12-mm-diameter glass coverslips and transfected with empty vector (pCAGGS), VP24 HA (pCAGGS), or VP24 HA mutants (pCAGGS) using Lipofectamine 2000® (LifeTechnologies). Twenty-four hours post-transfection, cells were serum starved for 4 h and then either mock treated or treated with 1,000 U/ml of human IFN-β for 30 min at 37°C. Cells were rinsed twice with PBS containing calcium chloride and magnesium chloride (PBS-CM), fixed with 4% parformaldehyde for 30 min, and blocked for 45 min at room temperature with 4% normal goat serum in PBS containing 0.5%BSA and 0.15% glycine (PBG). Subsequently, coverslips were incubated with rabbit anti-STAT1 (5 μg/ml; Santa Cruz) and mouse anti-HA (1:200) for 1 hr at RT. Coverslips were rinsed and incubated with Alexa 488-conjugated goat antibody raised against rabbit IgG, Alexa 555-conjugated antibody raised against mouse IgG, and Hoechst 33342 (Invitrogen). Images were acquired using an AxioPlan2 fluorescent microscope.