Fig. 3. Ectopic expression and phosphorylation of the Op18 N-terminus fused to a non-related -helical region. (A) KA8 cells were transfected with 12 µg DNA of each of the indicated pMEP4 derivatives, and hygromycin-resistant cell lines were selected and induced for the indicated time period (0 hours, 6 hours and 24 hours) with Cd²⁺ (0.5 µM). Expression levels were determined as described in Materials and Methods and expressed as a ratio relative to endogenous Op18 (the endogenous Op18 level in KA8 cells is about 10 µM). The mean of two independent transfection experiments is shown. (B) KA8 transfected with the indicated pMEP4 derivative were Cd²⁺ induced for 24 hours in the presence of the MT-disrupting drug nocodazole (0.5 µM), and phosphorylation stoichiometry of the expressed NR-derivatives was determined by native PAGE. Non-P Op18(1-57)-NR-helix correspond to non-phosphorylated E. coli-produced protein.