Journal: International Journal of Molecular Sciences

Article Title: CD93 Signaling via Rho Proteins Drives Cytoskeletal Remodeling in Spreading Endothelial Cells

doi: 10.3390/ijms222212417

Figure Lengend Snippet: CD93 triggers the pY774-Cbl-dependent signaling pathway in spreading ECs. ( a ) Cell lysates from early spreading HUVECs pretreated for 30 min with vehicle alone (DMSO) or 10 μM PP2 were analyzed by Western blotting using antibodies against pY774-Cbl and pY418-Src to confirm PP2 activity. Equal loading was confirmed by using anti-Cbl and anti-Src antibodies. ( b ) Quantification of pY774-Cbl phosphorylation levels from independent experiments performed as in ( a ). Values, normalized to Cbl protein levels, represent the percentage of pY774-Cbl levels relative to vehicle-treated cells (DMSO). *** p < 0.001; paired t -test. ( c , d ) HUVECs were transduced with lentiviral particles expressing unrelated (sh-unr) or CD93 shRNA (sh-CD93). Cells were detached from the plate, resuspended in complete growth medium, plated on the substrate, and fixed at early phases of spreading. Cells were analyzed by immunofluorescence using phalloidin, anti-CD93 antibody, and antibodies against pY774-Cbl ( c ) and Crk ( d ). White dot colocalization (wdc) images are shown as indicated. In the wdc pictures, magnification of the squared areas is shown (2.5×). Scale bars, 20 μm. ( e , f ) Quantification of cellular pY774-Cbl ( e ) and Crk ( f ) in the spreading border area (5 μm from the cell edge) of HUVECs transduced and treated as in ( c ) ( n = 14 cells for sh-unr and n = 16 cells for sh-CD93) and ( d ) ( n = 15 cells for sh-unr and n = 14 cells for sh-CD93). Data are presented as scatter plots and the fluorescence intensity of the pY774-Cbl +ve and Crk +ve signals per area is reported as arbitrary units (AU). * p < 0.05; Student t -test ( e ). *** p < 0.001; Mann–Whitney test ( f ).

Article Snippet: The Src family tyrosine kinase inhibitor PP2 was purchased from Merck KGaA.

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Transduction, Expressing, shRNA, Immunofluorescence, Fluorescence, MANN-WHITNEY