Supplementary Figure 13: The Mammalian Dcp2 Homolog Is Phosphorylated by MTOR, Related to Fig. 5.

(a) Schematic of the identification of phosphorylated Ser249 of DCP2 by mass spectroscopy. The peptides are represented by green lines and the phosphorylation site is indicated in purple. (b) Differential phosphorylation of DCP2 after treatment with rapamcyin assayed by targeted ion mass spectrometry. (c) SDS-PAGE of recombinant DCP2-MBP fusion protein purified by amylose-agarose affinity chromatography. (d) Ex vivo phosphorylation of DCP2 by MTOR. MTOR was immunoprecipitated from HeLa cells using an anti-MTOR antibody, washed, then incubated with 100 ng DCP2-MBP at 30 °C for the indicated times, and subjected to western blot using a rabbit DCP2-pSer249 affinity-purified antibody. Lower panel, density quantification of pSer249 in n = 3 independent experiments. Bar + / − SD. (e) DCP2-MBP phosphorylated with MTOR for 20 min as in in Supplementary Fig. 3d, then subjected to treatment (+) or no treatment (−) with calf intestinal alkaline phosphatase (CIAP) before western blot using antigen-purified anti-DCP2-pSer249 antibody.