Increased Susceptibility to Cortical Spreading Depression in the Mouse Model of Familial Hemiplegic Migraine Type 2
Total RNA and protein samples were isolated from brain of wild type (+/+), Atp1a2+/R887 (+/R887) and homozygous Atp1a2R887/R887 (R887/R887) mice at E19.5. A. Left panel. Semi quantitative Atp1a2 RT-PCR (254 bp fragment) on brain cDNA. The b-actin fragment (610 bp) is included as in-tube normalizer. Right panel, same Atp1a2 RT-PCR fragments digested by MspI. B. Protein blot of microsomal fraction probed with anti-α2 Na,K-ATPase antibody and anti-neogenin as loading control; the α2 Na,K-ATPase and neogenin bands appear at the expected size of 110 kDa and 52 kDa, respectively. C. Total brain lysates from adult wild type and Atp1a2+/R887 mice probed with anti-α1, α2, and α3 Na,K-ATPase antibodies; anti- tubulin as loading control. Densitometric quantization shows a 50% reduction of the heterozygous mutant α2 level compared to wild type. Error bars represent ± SD; Student's t test p<0.05, n = 3; α1, α2, and α3 Na,K-ATPases migrate as single bands according to the expected size of 110 kDa. D. Region specific immunoblot from cortex, cerebellum and total brain of adult wild type and Atp1a2+/R887 mice probed with anti-α2 and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (Figure 2D). Densitometry evaluation shows significant reduction in α2 level (p<0.05) of 50%, 35% and 40% in cortex, cerebellum and total brain, respectively. Error bars represent ± SD; Student's t test p<0.05, n = 3.