FIG. 5.

(A) SWP1 mRNA is upregulated during intracellular development. HFF monolayers were infected with E. cuniculi, and mRNA was isolated 24, 48, and 72 h postinfection. The volume of the 48-h sample was diluted 8-fold, and that of the 72-h sample was diluted 20-fold, to normalize for tubulin transcript. RT-PCR was performed with tubulin and SWP1-specific primer pairs on two cDNA amounts. PCR products were blotted onto nylon membranes and hybridized with digoxigenin-labeled tubulin or SWP1 cDNA fragments. The level of SWP1 mRNA was strongly upregulated from 24 to 48 h and moderately increased from 48 to 72 h postinfection. (B) Densitometric quantification of SWP1 RT-PCR products. Densitometric analysis of RT-PCR film exposures was performed using the BioDocII video documentation system (Biometra). Peak areas for SWP1 and tubulin were determined with the ScanPack 3.0 software (Biometra). The ratio of the SWP1 to the tubulin peak area was calculated for each time point of the kinetics; these ratios are expressed in arbitrary units. The amount of SWP1 mRNA, normalized to that of tubulin, increases fivefold from 24 to 48 h and more than sevenfold from 24 to 72 h postinfection.