FIG. 4.
(A) Electrophoretic profiles of infected and uninfected stained with Coomassie blue. HEp-2 cells were either mock infected or infected at an MOI of 5.0, and equal amounts of total protein, as determined by a modified Bradford assay, were resolved electrophoretically on an SDS-polyacrylamide gel and stained with Coomassie blue. Amounts of protein equivalent to that in each lane were extracted in the experiments shown in panel B. (B) Equal amounts of total protein from mock- and wild-type-infected lysates were harvested at 16 h postinfection and extracted with buffers containing 0 mM NaCl, 500 mM NaCl, or 2 M NaCl as described in Materials and Methods. Denatured soluble and insoluble fractions from the various buffer treatments were separated on an SDS-8% polyacrylamide gel and transferred to nitrocellulose, and the membrane was probed with lamin A/C antiserum. Bound anti-lamin antibody was detected with goat anti-mouse alkaline phosphatase-conjugated secondary antibody, followed by incubation with ECF substrate. The membrane was then scanned with a Molecular Dynamics Storm 860 PhosphorImager. Total counts were quantified with ImageQuant software. To determine the relative fractionation of UL31 proteins in these extractions, the membrane was blocked overnight with 5% nonfat dry milk in sterile PBS and then reprobed with UL31 protein-specific antiserum. UL31 protein was detected with goat anti-rabbit alkaline phosphatase-conjugated antiserum and chromogenic substrates. The displayed image was generated by scanning with a digital scanner.