Figure 3. Phosphorylation analysis of Dcp1a mutants in vivo and in vitro.

(A) The Dcp1a mutants S315A/S319A (double mutant), S315A, and S319A were co-transfected into HEK 293T cells with HA-tagged CA MAPKK1 for an in vivo phosphorylation assay. Protein samples were analyzed by western blotting using anti-Flag, anti-HA, and anti-tubulin. (B) In vitro phosphorylation assay of recombinant Dcp1a by ERK2. The recombinant wild-type and mutant Dcp1a proteins purified from E. coli were incubated with active ERK2 and [³²P]ATP at 30°C for 30 min. The reaction mixtures were separated by SDS-PAGE and analyzed by autoradiography. The recombinant proteins used in the reaction were detected by western blotting with anti-Flag or Coomassie blue (C.B.) staining. (C) Western blotting analysis of 3T3-L1 proteins using anti-p-Ser315. The samples from Fig. 1D were analyzed with anti-p-Ser315 and anti-Tubulin. (D) p-Ser315 detection in over-expressed Dcp1a. Flag-tagged Dcp1a and HA-tagged MAPKK1 mutants (CA or DN) were co-expressed in HEK 293T cells. Cells were treated in the presence or absence of 10 µM U0126 for 30 min, and cell extracts were isolated for western blotting with anti-p-Ser315, anti-Flag, and anti-tubulin. One representative of three to five independent experiments with similar results is shown.