figs3.

IGenotyping and expression analysis of adropin knockout and transgenic mice. A, An agarose gel electrophoresis shows PCR products for wild-type (Enho^+/+; 72 bp), heterozygous (Enho^+/-; 72 and 170 bp), and homozygous (Enho^+/-; 170 bp) genotypes performed on tail tissue genomic DNA using specific Enho wild-type and excised primers. B, Adropin monoclonal antibody is highly specific. Full western blot clearly shows a single band appearing at the right molecular weight of adropin (17 kDa) but not in homogenates from adropin knockout (Enho^-/-) mice. C, Genotyping of adropin transgenic mice performed on tail tissue genomic DNA by PCR using primers from the human β-actin promoter and Enho gene coding region, where the appearance of the 80 bp amplicon confirms the presence of the transgene. D, E, Quantitative RT-PCR, and western blot confirm that Enho mRNA expression (D) and protein levels (E) in AdrTg mice are significantly upregulated in the brain compared to the corresponding wild-type littermates. Unpaired t-test, **P<0.01, ***P<0.001. n=3 per group for qRT-PCR, andn=7 per group for western blot.