FIG 4.
The stability of NP is not affected by sumoylation. (A) 293T cells were transfected with phd or phdK33R and NP or NPK4,7R. At 24 h posttransfection, the cells were treated with 100 μg/ml CHX for the times indicated. The phd or NP was immunoblotted (IB) with anti-HA polyclonal antibodies. Immunoblot assay of whole-cell lysates with anti-actin antibodies are shown as loading controls (Input). (B) 293T cells were transfected with Ubc9 and Cer-SUMO1 together with NP or the NPK4,7R mutant form. At 36 h after transfection, the cells were treated with 100 μg/ml CHX for the times indicated. The NPs were immunoprecipitated (IP) with anti-HA–agarose, and the precipitated proteins were further analyzed by SDS-PAGE and immunoblotting with anti-HA polyclonal antibodies to detect NP. Western blot assays of whole-cell lysates with anti-actin antibodies are shown as loading controls (Input). The values to the left of the blots are molecular sizes in kilodaltons.