FIG. 6.

Regulation of AdipoR1 translation through its 3′UTR during muscle differentiation. A: Schematic representation of the generated construct containing the human AdipoR1 3′UTR fused downstream to the firefly luciferase gene (LUC-R1–3′UTR). B: Luciferase activity of LUC-R1–3′UTR construct in C2C12 myoblasts and myotubes; firefly luciferase activities were assessed and normalized to Renilla values as a control for transfection efficiencies. Activity of LUC-R1–3′UTR construct was normalized to activity of pGL3 control vector transfected in parallel plates. The results are expressed relative to LUC-R1–3′UTR activity in myoblasts. C: RT-PCR analysis to detect possible exon inclusion in the mouse AdipoR1 5′UTR in C2C12 myoblasts and myotubes. The PCR was performed using primers designed from exon 1 and exon 2 of the mouse AdipoR1 mRNA. D: qPCR analysis of AdipoR1 mRNA expression normalized to 18S rRNA in C2C12 myoblasts and 4 day–differentiated myotubes. E: Western blot analysis of mouse AdipoR1 protein expression in C2C12 myoblasts and myotubes. Equal amounts (70 μg) of protein lysates were resolved by means of 10% SDS-PAGE and were subjected to Western immunoblotting using anti-AdipoR1, anti-MyHC (as differentiation marker), and anti-tubulin (as loading control) antibodies. Data are expressed as the means ± SD of three independent experiments. P values were evaluated using Student t test. *P < 0.001.