Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Trogocytosis Results in Sustained Intracellular Signaling in CD4 + T Cells

doi: 10.4049/jimmunol.1201507

Figure Lengend Snippet: TCR-proximal signaling is sustained by trogocytosed molecules in trog+ cells. (A) Top panel, T cells were incubated for 30 min after recovery from standard trogocytosis assay before fixation and staining. In untreated cells, pZAP-70 (red) and the TCR (blue) colocalize with the trogocytosed MHC:peptide on the T cell surface. Bottom panel, The TCR and MHC:peptide colocalize on T cells treated with the Src inhibitor PP2 following the 30-min incubation period, but the there is minimal phospho-ZAP-70 detected. Middle panel, For cells with a 10-min PP2 treatment to extinguish signaling and before incubation for 20 min after PP2 removal, the pZAP-70 level rebounds and it colocalizes with the trogocytosed MHC:peptide and TCR. More than 100 individual T cell were imaged using a ×60 objective. Left image for each group is a fluorescence composite image to show the position of the T cell. Scale bars, 10 μm. (B) Integrated intensity of pZAP70 ≥6-fold above background in images as in (A) (top panels). Thirty-minute untreated T cells (green bar), T cells incubated with PP2 for 30 min (orange bar), or for treated for 10 min then incubated for 20 min after removal of PP2 (purple bar) are shown. (C) The frequency of pZAP-70 colocalizing with trogocytosed MHC:peptide for each treatment group is shown. (D) The frequency of TCR colocalizing with the TCR. Bars represent mean ± SEM. Horizontal lines indicate statistical comparison between indicated groups, *p ≤ 0.05.

Article Snippet: Cells incubated at very low density (10 4 /ml) were treated for 10 or 30 min with Src family tyrosine kinase inhibitor PP2 (Life Technologies) ( 61 ).

Techniques: Incubation, Trogocytosis Assay, Staining, Fluorescence

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

Journal: Oncotarget

Article Title: CD93 and dystroglycan cooperation in human endothelial cell adhesion and migration

doi: 10.18632/oncotarget.7136

Figure Lengend Snippet: ( A ) The schematic diagram illustrates the domains of CD93 and the 47-amino acid sequence of its cytoplasmic tail containing tyrosine 628 and 644. CTLD, C-type lectin-like domain; EGF-like, Epidermal Growth Factor repeats; Mucin. Mucin-like domain; TM, transmembrane domain. ( B ) Cell extracts from ECs spreading and growing on gelatin or laminin were immunoprecipitated with anti-CD93 antibodies. Immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine and anti-CD93 antibodies to confirm equal loading. ( C ) HUVEC were infected with a lentiviral vector expressing unrelated (unr) or DG (clones C7 or C10) shRNAs. Not infected ECs were also analyzed (HUVEC). Cell extracts from cells spreading and growing on laminin were immunoprecipitated or not (+, −) with anti-CD93 antibodies and analyzed by Western blotting with anti-phosphotyrosine antibodies. To confirm equal loading, whole cell lysates were analyzed by Western blotting with anti-CD93 and anti-β-actin antibodies. ( D ) HUVEC were allowed to adhere and grow on laminin in the presence (+) or not (−) of PP2 (10 μm). Cell lysates were immunoprecipitated with anti-CD93 antibodies and analyzed by immunoblotting with anti-phosphotyrosine and anti-CD93 antibodies to confirm equal loading. In the same cell lysates, phosphorylation on tyrosine 416 of Src, a protein modification that is closely correlated with kinase activity, was analyzed by Western blotting with anti-phospho-Y416 Src and anti-Src antibodies to confirm equal loading. All experiments were performed three-four times. ( E ) Human Lenti-X 293T cells, which do not express wild type CD93, were transiently cotransfected with a construct expressing human CD93 and the constitutively active (DP) or kinase dead (DN) Src kinase. Transfection with an empty vector (mock) is indicated. Cell lysates were immunoprecipitated with anti-CD93 antibodies and analyzed by Western blotting with anti-phosphotyrosine and anti-CD93 antibodies to confirm equal loading.

Article Snippet: The Src family tyrosine kinase inhibitor PP2 was purchased from Calbiochem (San Diego, CA, USA).

Techniques: Sequencing, Immunoprecipitation, Western Blot, Infection, Plasmid Preparation, Expressing, Clone Assay, Phospho-proteomics, Modification, Activity Assay, Construct, Transfection