Figure 5

Analysis of IGF1 expression levels in the genetically modified mouse model. (a) The strategy for Southern blot analysis of incorporation of the MLC enhancer in GM mice is the same as that shown in Fig. 2c. Genomic DNA of heterozygous GM mice and WT control was digested with HindIII and then hybridized with the 3′ external probe. The expected fragment sizes are: WT, 5.9 kb; GM, 3.7 kb. (b) Real-time PCR analysis of Igf1 mRNA levels in the gastrocnemius muscle of two-month-old GM mice and WT littermates. (n = 11–13 per group). (c) ELISA analysis of total IGF1 protein levels in the gastrocnemius muscle of two-month-old GM mice and WT littermates. The total IGF1 protein levels were normalized to the total protein levels. (n = 10–13 per group). (d) Western blot analysis of the Akt phosphorylation levels in the gastrocnemius muscle of one-month-old female GM mice and WT littermates. Antibodies against phosphorylated Akt (Ser473), Akt (Thr308), and total Akt were used. GAPDH was used as a loading control. The ratio of phosphorylated Akt (Ser473)/Pan-Akt and phosphorylated Akt (Thr308)/Pan-Akt were determined by calculating the intensities of the blots and are shown below. All gels/blots were run under the same experimental conditions. Shown are cropped gels/blots (Full-length gels/blots with indicated cropping lines are shown in Supplementary Figure S10). Bars depict mean values, and error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.0001, ****P < 0.000001. Statistical significances were analysed by Student’s t-test.