Figure 1.
JNK activators affect localization of DCP1a to P bodies. (A) HEK293IL-1R cells were transfected with vectors encoding GFP-DCP1a together with JNK activators (MEKK1 or a TAK1-TAB1 fusion protein) or with JNK wild type or a catalytically inactive mutant (JNK2K55R). Colocalization of GFP-DCP1a (green) with JNK (red) was analyzed by fluorescence microscopy. The fraction of transfected cells exhibiting a phenotype identical to that shown in the representative image was determined by scoring ≥50 cells from three to four independent transfections. Bar, 10 µm. IF, indirect immunofluorescence. (B) HEK293IL-1R cells were transfected in parallel cultures, and modifications of GFP-DCP1a were analyzed by mobility shifts (labeled 1–3) upon SDS-PAGE and subsequent Western blot analysis. Equal expression and activity of cotransfected constructs were validated by anti-JNK, anti-GFP, anti-TAK1, and antiphospho–c-Jun (P-c-Jun) antibodies. Proteins from the same samples were separated on three gels and transferred to three membranes (M1–M3), which were probed with the indicated antibodies. Ponceau staining or hybridization with antiactin or antitubulin antibodies is shown to indicate equal protein loading. IB, immunoblot; n.s., nonspecifically detected protein band that served as a loading control.