Expression of poMx1 in double-transgenic Vero cells examined by fluorescence microscopy and immunoblotting (clone V50). Transgenic cells were induced with 1 μg/mL doxycycline for 24 h. Induced mono-transgenic cells (clone Vero Tet/R1) are presented for control purposes (low background and absence of cross-reactivity with endogenous Cercopithecus aethiops Mx). Clone V50 combined less than 5% spontaneous expression with more than 95% expression and intense cytoplasmic granular staining upon exposure to doxycycline. The Western blot reveals a specific band in the lane for induced cells. The molecular mass corresponding to this band is compatible with that of poMx1 (74 kDa). For indirect immunofluorescence (A), cells were permeabilized and stained by sequential incubation with a rabbit antiserum raised against human MxA (a gift from I. Julkunen, Helsinki, Finland), which had been shown to cross-react with porcine Mx1 [24], and with Alexa 488-conjugated goat anti-rabbit IgG. (B) Immunoblots of 4–12% sodium dodecyl sulfate polyacrylamide gels with total cell proteins extracted from induced (dox +) and non-induced (−) V50 cells (10 μg total protein per lane, as determined by a micro-BCA assay). Immunostaining was done by sequential incubation with a cocktail of the above-mentioned rabbit antiserum and anti-β-actin mAb and a mix of HRP-conjugated anti-rabbit and anti-mouse IgG. Blots were developed by incubation with 3-amino-9-ethylcarbazole. The positions of protein size markers in kilodaltons are indicated (MW). (A color version of this figure is available on line at www.vetres.org.)