PGE₂ signaling kinetics. (A) Immunoblot of CD3⁺ T lymphocytes stimulated with PGE₂ (10μM) over a 60-minute time course with the use of a phospho-PKA substrate antibody (RRXpS/pT). (B) Relative density of 4 independent immunoblots plus SEM as depicted in panel A. (C) Mass spectrometry work flow. Three samples from 1 blood donor (CT, control; 10 and 60 minutes of PGE₂ [10μM] stimulated) were lysed separately and in-solution trypsin digested. The resulting peptides were labeled with stable isotope labeling by reductive amination. Subsequently, the 3 samples of the 3 different time points were mixed at a ratio of 1:1:1. After SCX fractionation, phosphopeptides were subjected to online TiO₂-LC enrichment, coupled to a LTQ Orbitrap mass spectrometer. Data analysis was conducted as described in the “Bioinformatic handling of mass spectrometry data.” LC-MS/MS indicates liquid chromatography tandem mass spectrometry.