FIG. 4.

HeLa and HeLa/SV5-V cells were or were not treated with siRNA or control RNA twice at 24-h intervals. At 100 h posttreatment, IFN was or was not added to the culture medium, and 20 h later the cells were metabolically labeled with [³⁵S]methionine for 5 h in the presence or absence of IFN as appropriate. Cells were then harvested, and the levels of DDB1 estimated by coimmunoprecipitation with V (a) (note that DDB1 was detected in HeLa/SV5-V cells treated with control RNA but not when they were treated with siRNAs and that DDB1 was not immunoprecipitated from HeLa cells as they do not express V) and immunoblot analysis (b). Levels of STAT1 in the cell samples were also examined by immunoprecipitation (c) and immunoblot analysis (d). Note the increase in levels of STAT1 in HeLa/SV5-V cells treated with siRNAs, especially following incubation with IFN (lanes 3 and 4), but not in cells treated with control RNA (lanes 5 and 6).