Deletion of the Sema4D calmodulin-binding domain causes constitutive shedding of the Sema4D exodomain. (A) Illustration of the Sema4D constructs that were transfected into Chinese hamster ovary (CHO) cells. D781Δ retains the calmodulin-binding domain; P761Δ does not. (B, top) Western blot of CHO cell supernate showing constitutive shedding of Sema4D. (B, middle) Relative surface expression of Sema4D in CHO cells detected by flow cytometry and normalized relative to wild-type (WT) controls. (B, bottom) A summary of three experiments. The relative fragment:Sema4D ratio is the ratio of the Sema4D fragment level versus surface Sema4D expression level normalized to that of the wild-type, which was set as 1.0. N.S., not significant; TM, transmembrane. *P < .05.