Phosphoproteomic data recapitulate canonical thrombin signaling. (A) Immunostaining for PECAM-1 (green) and VWF (red) of SILAC BOECs treated with 1 U/mL thrombin for the indicated time. The bottom panels show zoomed-in areas (boxed) in the top panels. Bar = 50 µm. LSM510 confocal laser scanning microscopy (Carl Zeiss), ×63/1.4 oil objective. (B) Three principal components that captured 67.8% of the total variance of the BOEC phosphoproteome changes during thrombin stimulation are shown. Principle component analysis was performed on the SILAC ratios of the phosphosites quantified in all experiments. Colors are as in (A). (C) Thrombin signaling according to Ingenuity. MS-identified phosphoproteins are shown in gray, and those for which significantly regulated phosphosites were measured are indicated with an orange border. Each P indicates a distinct regulated phosphosite, reported in (D). (D) Detailed list of the phosphosites depicted in (C). Bold, functional phosphosite (according to PhosphoSitePlus); ^, redundant site. The reported amino acid position refers to the protein within the protein group for which a reference number (referred to as UniProtKB) was found in the PhosphoSitePlus database. (E) Mean and standard deviation for the MS-based quantifications. Western blot analysis for phospho mitogen-activated protein kinase (MAPK)1/ERK2 (Y187), phospho MAPK3/ERK1 (Y204), phospho MAPK14/p38 MAPK (T180/Y182), and phospho MYL/MLC (T19/S20). Time points are as indicated in (A). α-tubulin was used as loading control.