Figure 5. I14 in mature LEP is essential for LEP-LEPR interaction and the downstream STAT3 pathway.

(A) Sanger-sequencing of the RT-PCR products showed that the Lep mRNA of Lep^{∆I14/∆I14} rat had a deletion of 3 nucleotides ATC encoding an Ile residue. (B) Western blot showed that the mature LEP^∆I14 protein is stably expressed in the WAT of Lep^{∆I14/∆I14} rats. Shown is one of three independent experiments. (C) ELISA showed that serum LEP^∆I14 in male Lep^{∆I14/∆I14} rats (n = 5) is significantly increased compared to that of serum LEP^WT in the male WT controls (n = 5). (D) Computer assimilation of LEP-LEPR interaction using available information from their human homologs: LEP (PDB number: 1AX8) and LEPR (PDB number: 3V6O). (E) STAT3 reporter assay. 293FT cells were treated with WT and mutant recombinant rat LEP proteins at different concentrations after transient transfection of pcDNA-Lepr, pRL-TK and pGL6-Stat3. Relative luciferase activity was determined by firefly luciferase light units normalized by that of renilla luciferase.