Stat3 and phospho stat3

For the analysis of individual proteins, tissues were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate) using a handheld, battery-operated homogenizer. 10 μg lysates were loaded per lane and electrophoresed on 10% SDS-polyacrylamide minigels with reducing, denaturing sample buffer. The separated proteins were transferred to PVDF membranes and probed with antibody O-17 (IBC) to the C-terminus of osteopontin and anti-HCAM to the cytoplasmic domain of CD44 (Santa Cruz) and to STAT3 and phospho-STAT3 (Cell Signaling Technology). Antitubulin serves as a loading control.

All analyses were performed as described previously [10 (link), 52 (link)]. For immunoblotting analysis, whole-cell lysates were made using a RIPA lysis buffer, resolved on Bis-Tris 10% gel, and transferred to PVDF membranes. The blots were then probed with various primary antibodies. E-cadherin, fibronectin, N-cadherin, alpha-catenin, and vimentin (BD Biosciences); Twist-1 and DJ-1 (Abcam); STAT3 and phospho-STAT3 (Cell Signaling Technology); HA and GFP (Santa Cruz Biotechnology); V5 (Life Technologies); β-actin (Sigma-Aldrich).
For Immunofluorescence staining, 1 × 10⁴ cells were incubated with specific primary antibodies, washed, and then incubated with secondary antibodies (Invitrogen). Images were acquired using a confocal microscope LSM 700 (ZEISS).
For immunohistochemistry, a tissue microarray slide (CSA) was purchased from Super Bio Chips, and the expression of TrkC at the tissue microarray slide was evaluated by using an indicated antibody. In addition, the evaluation of immunostaining intensity was performed and obtained the score by ImageJ software.

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.