The transfected MEK/ERK chimera increases Sp1 phosphorylation in HeLa cells, whereas JNK^DN causes its dephosphorylation in PC3 cells. HeLa and PC3 cells were transfected with the constitutively active MEK/ERK chimera (MEK/ERK^A; see Figure 2) or with JNK^DN, respectively. Equal amounts of nuclear extract from transfected and untransfected cells were treated (or not) with alkaline phosphatase prior to fractionation on a 10% polyacrylamide–sodium dodecyl sulfate (polyacrylamide-SDS) gel and transfer to a nylon membrane. The Western blots were overlain with anti-Sp1 polyclonal antibodies that recognize both the phosphorylated and unphosphorylated forms of the transcription factor. Transfection of HeLa cells with the active MEK/ERK chimera induced phosphorylation of Sp1 (A), whereas transfection of PC3 cells with the dominant negative form of JNK abolished it (B).