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Journal: bioRxiv
Article Title: Scube2 Modulates Coronary Vessel Formation during Cardiac Growth and Regeneration in Zebrafish
doi: 10.64898/2025.12.17.695032
Figure Lengend Snippet: (A) SCUBE2 interacts with the PDGF-B ligand. HEK-293T cells were transfected with FLAG-tagged SCUBE2 (FLAG.SCUBE2) and His-tagged PDGF-B (His.PDGF-B) individually or in combination for two days. Cell lysates were subjected to immunoprecipitation (IP) using an anti-His antibody, and the co-immunoprecipitated SCUBE2 protein was detected by Western blot (WB) analysis using an anti-FLAG antibody (upper panel). The expression of FLAG.SCUBE2 and His.PDGF-B was confirmed by anti-FLAG and anti-His WB analysis, respectively (middle and lower panels). (B) SCUBE2 is capable of binding PDGFRβ. HEK-293T cells were transfected with FLAG.SCUBE2 and HA-tagged PDGFRβ (HA.PDGFRβ) either individually or together for two days. Lysates were immunoprecipitated using an anti-FLAG antibody, and the associated PDGFRβ protein was detected by WB using an anti-HA antibody (upper panel). The expression of FLAG.SCUBE2 and HA.PDGFRβ was validated by anti-FLAG and anti-HA WB analysis, respectively (middle and lower panels). (C) SCUBE2 promotes PDGF-BB-induced PDGFRβ signaling. HEK-293T cells were co-transfected with vector control and FLAG.SCUBE2, or with HA.PDGFRβ and FLAG.SCUBE2, for two days. PDGFRβ signaling was then stimulated by the addition of recombinant PDGF-BB protein for 30 minutes at the indicated concentrations. The levels of phosphorylated PDGFRβ (p-PDGFRβ) as indication of ligand-induced signaling, total protein of PDGFRβ, and SCUBE2 were analyzed by WB using anti-p-PDGFRβ, anti-PDGFRβ, and anti-FLAG antibodies, respectively. Protein levels of p-PDGFRβ and total PDGFRβ were quantified and plotted as a line graph (D). Statistical comparison was performed between the SCUBE2 + PDGFRβ and vector + PDGFRβ groups. Data are presented as mean ± S.D. from four independent experiments. *, P < 0.05.
Article Snippet: The His-tagged PDGF-B (catalog HG10572-NH) and
Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Binding Assay, Plasmid Preparation, Control, Recombinant, Comparison