Northern blot and in situ hybridizations indicate that the unc-79 mRNA is widely expressed throughout the central nervous system, and as expected there is a modest reduction in mRNA in homozygous mutant P0 brain tissue, presumably due to nonsense mediated decay (Figure S1). To determine whether a full-length unc-79 protein was produced in mutants, we generated polyclonal antibodies against a C-terminal portion of the protein (see Figure 3C). Immunoblot analysis of P0 whole brain tissue lysates from wild-type and homozygous mutant animals indicates an absence of the expected band at ∼290 kDa in mutant versus wild-type homogenates (Figure 3D). In invertebrates, null alleles of the unc-79 gene dramatically influence expression of the NALCN protein homolog [10]–[12]. Western blot analysis of NALCN expression in postnatal day 0 (P0) wild-type and Lwt/Lwt littermates did not reveal any obvious differences in expression (Figure S2).
