DPT binds to ITGA4 and reduces hematopoietic cell adhesion to VCAM1. (A) Schema of the far western blot strategy. Recombinant dermatopontin (rDPT) was separated by electrophoresis and transferred to PVDF membrane, followed by incubation with endothelial cell whole cell lysate. A binding partner specific antibody (Ab) was used for detection of potential binding partner. (B) Far western blotting shows ITGA4 binding to immobilized rDPT (left panel). The control (CTL) lane incubated without any primary antibody. Quantification of the anti-ITGA4 lane pixel values after scanning densitometry, using ImageJ and the plot profiler function (right panel). (C) Far western blotting using several integrin family antibodies to determine their potential interaction with rDPT. (D) 4′,6-diamidino-2-phenylindole (DAPI)-stained lin⁻ bone marrow cells binding to VCAM1-coated wells, with or without 1 µg/mL DPT. (E) Quantification of lin⁻ cells bound to VCAM1-coated wells was performed using ImageJ. Included are experiments in which anti–VLA-4 was used to inhibit cell adhesion as a control. (F) DAPI-stained lin⁻ bone marrow cell binding to VCAM1-coated wells in the presence of 4 µg/mL DP-4 or DP-S (scrambled) peptide. (G) Quantification of images shown in (F). The bar graphs show the results of 3 independent experiments. Scale bars indicate 100 μm. All data are shown as means with standard deviations. The P values were derived using a Student's t-test. PVDF, polyvinylidene difluoride.