Journal: iScience

Article Title: Ligand preference of EphB2 receptor is selectively regulated by N-glycosylation

doi: 10.1016/j.isci.2025.112386

Figure Lengend Snippet: The glycosylation of EphB2 is indispensable for ephrin-A5/B2 binding specificity (A) Top: western blot and Coomassie brilliant blue staining results of purified EphB2 protein before/after PNGase F treatment. Bottom: western blot results of endogenous EphB2 from adult and P10 mice hippocampus before/after PNGase F enzyme treatment. GAPDH served as a loading control. (B) Flow diagram of FCM assay. EphB2-expressing cells bound by pre-clustered ephrin-A5 or ephrin-B2 were recorded by flow cytometry and translated to the relative MESF values. (C) The FCM results are analyzed by the “one-site specific binding” model to fit the saturation binding curves and obtain the K D values. The K D of EphB2 WT to ephrin-B2 and ephrin-A5 are 3.526 nM and 111.9 nM; the K D of EphB2 4Q to ephrin-B2 and ephrin-A5 are 29.91 nM and 32.29 nM. (D and E) Representative confocal microscopy images of ligand-induced ligand/receptor colocalization assays upon stimulating with ephrin ligands. Green represents cells expressing EphB2 4Q, and red represents 647-labeled pre-clustered ephrin ligands. Scale bar: 10 μm. Averaged ratios of cells with EphB2 4Q/ephrins colocalization after stimulation are plotted as (E). Statistical significance was performed by two-way ANOVA; Tukey’s post-hoc multiple comparisons test; ns, not significant; ( n = 3). (F) Representative western blot results of immunoprecipitated C-terminal 3 × FLAG EphB2 WT or EphB2 4Q in HEK293T cells after ephrin-A5 or ephrin-B2 stimulation. (G) Measurement of relative EphB2 tyrosine phosphorylation, correlated with (F). Statistical significance was performed by one-way ANOVA; Tukey’s post-hoc multiple comparisons test, ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ( n = 3). All data are represented as mean ± SD.

Article Snippet: Quantumtm MESF microsphere kits , Bangs Laboratories , PSD818.

Techniques: Glycoproteomics, Binding Assay, Western Blot, Staining, Purification, Control, Expressing, Flow Cytometry, Confocal Microscopy, Labeling, Immunoprecipitation, Phospho-proteomics

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet:

Article Snippet: QuantumTM Alexa Fluor® 647 MESF kit , Bangs Laboratories , Cat# 647.

Techniques: FLAG-tag, Control, Virus, Generated, Subcloning, Recombinant, Staining, Flow Cytometry, Selection, Gene Knockout, Plasmid Preparation, Cloning, Expressing, Software