EphB2-Fc stimulation of wild type, p.P54L, p.T111I ephrin-B1 expressing NIH 3T3 cells. (A) NIH 3T3 cells expressing wild type and p.T111I ephrin-B1 were detected by fluorescent microscopy as cell clusters after the EphB2-Fc stimulation (indicated by arrows). Cells expressing p.P54L were scattered like the cells in the control (Fc treatment). (B) Following a time course of 5 to 30 min of EphB2-Fc stimulation, Western blot analysis of wild type, p.P54L and p.T111I ephrin-B1 expressing NIH 3T3 cells using phospho-ephrin-B (Tyr324/329) antibody and anti-ephrin-B1 antibody was performed. Cells transfected with wild type ephrin-B1 and treated with Fc only were used as a control. The anti-ephrin-B1 antibody (A-20) was used to demonstrate the whole amount of the ephrin-B1 protein loaded. Differences in the amount of ephrin-B1 on the Western blots can be explained by unequal amounts of ephrin-B1 in the lysates. This may be due to differences in the expression from the transiently transfected EFNB1 cDNA plasmids. Protein sizes were determined using Precision Plus Protein™ Standards Dual Color (BIO-RAD).