The mediobasal hypothalamus was isolated with a brain dissection block, as previously described [27 (link),29 (link),30 (link),31 (link)]. Tissues were homogenized using a TissueLyser II (Qiagen, Tokyo, Japan) in fresh RIPA buffer (containing 200 mMTris/HCl (pH 7.4), 130 mM NaCl, 10%(v/v) glycerol, 0.1%(v/v) sodium–dodecyl sulfate (SDS), 1%(v/v) Triton X-100, 10 mM MgCl2) containing anti-proteases and anti-phosphatases (Sigma-Aldrich, St. Louis, MO, USA). The tissue lysates were centrifuged for 30 min at 18,000 g in a microfuge at 4 °C. The mediobasal hypothalamus total protein lysates were subjected to SDS-polyacrylamide gels (SDS–PAGE), then electrotransferred on a PVDF membrane and probed successively with the following antibodies: Phospho-PI3K P85, PI3K P85, phospho-STAT3 and STAT3 (Cell Signaling, Danvers, MA, USA); β-actin (Sigma-Aldrich, St. Louis, MO, USA) after blocking the membranes with 5% BSA blocking buffer. For the protein detection, we used horseradish-peroxidase-conjugated secondary antibodies (Dako Denmark, Glostrup, Denmark). Specific antigen-antibody bindings were visualized using chemiluminescence method according to the manufacturer´s instructions (Pierce ECL Western Blotting Sustrate, Thermo Fisher Scientific, Grand Island, NY, USA). Values were expressed in relation to β-actin.
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