Rabbit anti β dg

Immunoprecipitation and immunoblotting experiments were performed as previously described [49 (link)]. For co-immunoprecipitation experiments cells were lysed in Co-ip buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, protease and phosphatase inhibitors, Sigma-Aldrich, St Louis, MO, USA). The following antibodies were used for immunoblotting: rabbit anti-CD93 (H190), mouse anti-phosphotyrosine, mouse anti-ubiquitin, and rabbit anti-β-DG (Santa Cruz Biotechnology); mouse anti-Cbl (Millipore); rabbit anti-phospho-Cbl(Y731), rabbit anti-phospho-Y416 Src, and rabbit anti-Src 32G6 (Cell Signaling Tech., Danvers, MA, USA); rabbit anti-phospho-Cbl (Y700) (Abcam, Cambridge, UK); mouse anti-β-actin (Sigma-Aldrich). For immunoprecipitations, mouse anti-CD93 antibodies (mAb 4E1) were used coupled to Dynabeads Pan Mouse IgG (Thermo Fisher Scientific). The Src family tyrosine kinase inhibitor PP2 was purchased from Calbiochem (San Diego, CA, USA).

Cells were seeded on glass coverslips, fixed in 3% paraformaldehyde, and then treated as previously described [43 (link)]. The primary antibodies used were: mouse monoclonal anti-CD93 (mAb 4E1, 0.6 μg/μL [5 (link)]) 1:25, rabbit anti-β-DG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-phospho-Cbl (Y774) (Millipore, Billerica, MA, USA). Fluorescent images were captured using a Leica TCS SP2 laser-scanning confocal microscope. The quantitative colocalization analyses of CD93 and β-DG or phospho-Cbl signals were performed on optical sections captured at cell/substrate adhesion sites using ImageJ and the JACoP plug-in to determine Manders' coefficient M₁ [44 (link)], which represents the percentage of CD93 pixels that overlap β-DG or phospho-Cbl pixels. To show colocalization events by white dots, images were generated using ImageJ and the Colocalization plug-in.