![Figure 1. 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.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/13/10.1182_blood-2010-01-266650/4/zh89991057590001.jpeg?Expires=1735840502&Signature=ETEMCqFLIarpolWmd5Ft-5-SfaMeFAwydtle8BRFO~W5c88gQy1J80iZq281xN1jNeEydPYpyq6CuOAB8ddSNAMreJHsHdY5th2DXuXByQRzWA9Tb5MXDKcENzIHSUaa7AhYgmAO8D8FyZyfzDuF9Mh4wc--3VIqSfrNnkEonq1n-1CrQO~5uIROgUSPyXpNCvMQHMiN2WZvhqj1EJi~JaU-QgW~QgAyKlEV8JCfM-6vk99gwrACTPCGdpAVXJxeSYltQENlCsi1kaoyqSKHPO~U9VL94kO5yyc8uJOHKDD8G5wOo9JRvhGRTFmav4uDu~naeg2uDTWs6CX3UAInNw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
