Figure 6.

Coupled in vitro kinase and phosphatase workflow and results. (A) Workflow for coupled in vitro kinase and phosphatase assays. Purified substrates were incubated with or without purified CK2 in in vitro kinase assay buffer at 28°C for 2 h and dialyzed overnight against in vitro kinase assay buffer without ATP. Substrates were then incubated with or without purified PP6 in in vitro phosphatase buffer at 28°C for 2 h, reduced, separated by SDS-PAGE, Coomassie stained, excised, digested with protease, and analyzed by LC-MS/MS. (B–D) Aligned reciprocal tandem mass spectra results for candidate CK2/PP6c substrates from in vivo (top, blue) and in vitro (bottom, purple) analyses. (E–G) Quantification of phosphorylation sites abundance in control, CK2 and CK2 and PP6 treated conditions using Students T-test, ^*p < 0.05, n = 2 independent experiments for EF1D, 3 for others.