Given the genetic mapping results, we compared the responses of mice that were heterozygous for the Dcc gene (on a C57BL/6 background) with those of their wild type (C57BL/6 J) littermates after chronic morphine treatment. Adult heterozygous Dcc mice were used because homozygous Dcc knockout mice die within a few hours of birth [30]. To determine whether adult Dcc heterozygous mice have reduced DCC protein levels we conducted immunoblot analysis of spinal cord homogenates and found that the Dcc heterozygous mice had ~50% lower DCC protein levels than wild-type littermates (Figure 3).
