FIG. 5.
(A) HEp-2 cells were transiently transfected with 4.5 μg of pJB233, a full-length UL31/pcDNA3 construct. Cells were fixed and permeabilized at 45 h posttransfection and incubated with UL31/GST-specific rabbit polyclonal antisera and lamin A/C monoclonal antibody. Bound antibodies were detected with Alexa 488 fluorophore-conjugated donkey anti-rabbit antibodies (green, UL31 protein) and Alexa 568 fluorophore-conjugated donkey anti-mouse antibodies (red, lamins A/C). Areas of colocalization of the proteins are indicated by a yellow signal in the merged image. The differential interference contrast (DIC) image shown is of the same transfected cell. Images were acquired with a 63× oil objective. (B) HEp-2 cells were transiently transfected with 4.5 μg of pJB234, a full-length UL34/pcDNA3 construct. Transfected cells were fixed and blocked with 10% BlockHen II (Aves Labs) and reacted with anti-UL34 chicken antibody (1:4,000) and lamin A/C monoclonal antibody. Bound antibodies were detected with Texas Red-conjugated donkey anti-chicken immunoglobulin and FITC-conjugated donkey anti-mouse immunoglobulin. Two different images of the cells are shown: the upper panel shows a cell scored as having cytoplasmic lamin A/C, whereas the lower panel shows a cell having nuclear lamin A/C. Cumulative results are presented in Table 3. (C) HEp-2 cells were cotransfected with 2.5 μg of pJB233 together with 2.5 μg of pJB234. At 45 h posttransfection, cells were fixed in ice-cold methanol for 20 min at −20°C and rinsed thoroughly in PBS. The fixed cells were blocked with human serum and incubated with UL31 protein-specific rabbit polyclonal antiserum, UL34 protein-specific chicken antiserum, and mouse anti-lamin A/C monoclonal antibody. Bound primary antibodies were detected with donkey anti-rabbit immunoglobulin conjugated with Alexa 488 fluorophore, donkey anti-chicken immunoglobulin conjugated with Alexa 568 fluorophore, and donkey anti-mouse immunoglobulin conjugated with indodicarbocyanine. Fluorescence and Nomarski differential interference contrast digital micrographs were collected with Fluoview software with a 63× oil objective. The lamin A/C-specific signal was pseudocolored blue for illustrative purposes.