(A) Ephrin-A5 is expressed at higher levels on EphA7-/- adult primary myogenic cells than it is on WT. (B) Mutations were made in the EphA7 and ephrin-A5 coding sequences of C2C12 myoblasts using double-nickase targeting, and single clones were expanded and analyzed by RT-PCR for inactivating mutations. Note: The red box in B indicates provisional sequencing of the targeted mutation in EphA7. (C) In high serum conditions, differentiation (indicated by expression of myogenin) is minimal in low density cultures of unedited, EphA7KO, and ephrin-A5KO C2C12 cells. Unedited C2C12 cells respond to high density by differentiating even in the presence of high serum, but EphA7KO and ephrin-A5KO C2C12 cells do not. At either low or high densities, unedited and EphA7KO C2C12 cells differentiate in high serum in response to exposure to EphA7-Fc, but ephrin-A5KO C2C12 cells do not. Table shows all significance comparisons by density, substrate, and genotype. (D) Unedited and EphA7KO C2C12 cells phosphorylate ERK1/2 following 10' exposure to soluble EphA7-Fc to a greater degree than do ephrin-A5KO C2C12 cells; all values were normalized to loading control. Error bars = SEM, p ≤ 0.01 (**), 0.001 (***) or 0.0001 (****). Scale bars = 50 μm.