Figure 5: Effects of treatment with rLECT2 on VEGF165-stimulated VEGFR2 tyrosine phosphorylation and downstream protein expression in HUVECs.
(a) Immunoblot of phosphorylation VEGFR in HUVECs. Serum-starved HUVECs were incubated with indicated treatment for 15 min. Cell extracts were subjected to immunoprecipitation (IP) with an antibody against the phosphotyrosine pY99. Precipitated proteins were analyzed via immunoblotting (IB) with an antibody against VEGFR2 (KDR) present in pY-VEGFR2. The same blots were subsequently reprobed with antibodies against VEGFR2 present in the receptor. (b) Immunoblot showing expression of the indicate proteins in HUVECs. HUVECs were serum-starved for 8 h and then treated with rLECT2 protein (5 nM) for 15 min prior to treatment with VEGF. (c) HUVECs were serum-starved for 8 h and then treated with rFc-Tag protein (5 nM) as negative control for 15 min prior to treatment with VEGF. Each treatment was performed in triplicate. (d) An in vitro binding assay to detect LECT2 and VEGFR2 binding. An Fc-tagged rLECT2 protein and His-tagged VEGFR2 extracellular domain (1–746 amino acids) were incubated and purified using a nickel-affinity column. The washed precipitates were then subjected to the western blot. (e) 293T cells were transfected with a control vector, HA-tagged LECT2 (LECT2-HA), or V5-tagged VEGFR2 (VEGFR2-V5) as indicated. Cell lysates were immunoprecipitated with an HA antibody and then subjected to immunoblotting with the indicated antibodies. (f) Endogenous interactions between LECT2 and VEGFR2 in HUVECs were evaluated. The HUVECs were treated with 293T cell-expressing control or LECT2 CM for 30 min, and cell lysates were harvested. HUVEC lysates were immunoprecipitated with an antibody as indicated.
