Endospanin1 is necessary for leptin-induced PI3K activity. (A–B) Representative western blot of 10 nM leptin-stimulated STAT3 (A) and AKT (B) phosphorylation following 2 consecutive transfections of HEK293-OBRb with Endo1 or control siRNA for 4 days. Densitometry analysis of (A) and (B) is presented on the right panel. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3–5). (C) Representative images showing phospho-AKT (Ser473) immunoreactivity in the ARC of mice 8 weeks after stereotactic injection with CTRL shRNA or Endo1 shRNA-expressing lentiviral vectors. CTRL-CD-CD or Endo1-CD-CD mice received for 15 min intraperitoneal injections of either vehicle (saline) or leptin (2 mg/kg). Densitometry analysis is presented on the right panel. Leptin increased the number of phospho-AKT (Ser473)-immunoreactive cells in the ARC, while Endo1 silencing significantly abrogated this effect. ns, p > 0.05; *, p < 0.05 (n = 3–6). Data are presented as means ± SEM.