Figure 2. Mutant Na⁺ channel mRNA and protein are expressed in mouse skeletal muscle tissue.

(A) Northern blots of total RNA extracted from pooled hind-limb skeletal muscle, quadriceps, gastrocnemius/soleus, heart, or brain tissues (5 μg loaded per lane; n = 3–8 mice per group). The 3′-UTR probe specific for mNa_V1.4 hybridized to an 8.5-kb full-length Na⁺ channel mRNA in both normal (+/+) and mutant (+/m) muscle samples but not those from heart or brain (top panel). The blot was rehybridized with a GAPDH probe (bottom panel). (B) RT-PCR of total RNA from (+/+) or (+/m) mouse muscle using primers c (exon 23) and b (exon 24). Digestion of the mutant RT-PCR product with HpaI (producing 0.89-kb and 0.54-kb fragments) or NspI (preserving a 0.71-kb fragment) demonstrated that approximately 30%–50% of transcripts in mutant muscle encoded the Met1592Val variant. The lane labeled “M” shows a 100-bp marker DNA ladder. (C) Western blot of membrane proteins isolated from (+/+), (+/m), or (m/m) mouse quadriceps muscle, heart (HT), and brain (BN) tissues (10 μg loaded per lane). The L/D3 monoclonal antibody specifically recognizes Na_V1.4 but not cardiac or brain Na⁺ channel isoforms.