Journal: Scientific Reports
Article Title: The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat
doi: 10.1038/srep28508
Figure Lengend Snippet: ( 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.
Article Snippet: Each Lep ∆ I14/ ∆ I14 rat was subcutaneously implanted an Alzet osmotic minipump (model 2001, Durect) filled with rat recombinant LEP (Genscript) at a dosage of 200 μg/kg/day (in sterile saline), while each WT rat was subcutaneously implanted an Alzet osmotic minipump (model 2001, Durect) filled with sterile saline as control.
Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Residue, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Reporter Assay, Mutagenesis, Recombinant, Transfection, Luciferase, Activity Assay