Figure 9.
Analysis of the growth rate of SV40 Pt mutant in Cos-7 cells by immunocytochemistry and Western blotting. (A) Cos-7 cells were transfected/infected with either SV40(776) WT or its Pt mutant as described in materials and methods and at 6th day posttransfection, cells were fixed in cold acetone, washed with 1 × PBS and incubated with both anti-agno polyclonal rabbit [19] and anti-VP1 (PAb597) monoclonal mouse primary antibodies (1:200 dilution each). After washing the primary antibodies with 1 × PBS, cells were incubated with secondary antibodies, goat anti-rabbit FITC-conjugated (green), and goat anti-mouse Rhodamine-conjugated (red) antibodies. Cells were finally washed with 1 × PBS, mounted in mounting media containing DAPI stain (Vector laboratories Inc. CA) and examined under fluorescent microscope. (B) Western blot analysis of nuclear extracts for VP1 expression, prepared from Cos-7 cells transfected/infected with either SV40 (776) WT or its Pt mutant as described for panel A above. Nuclear extracts (10 μg/each sample) were resolved on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane and probed with anti-VP1 monoclonal antibody (PAb597). In lane 1, nuclear extracts from untransfected Cos-7 cells were loaded as a negative control (- Cont.).