Sortilin

Western blotting was performed with specific antibodies against phospho-p38MAPK-T180/Y182 (#4511), or phospho-AKT-T308 (#2965) from Cell Signaling, or phospho-ERK1(T202/Y204)/ERK2(T185/Y187) (R&D AF1018) and ERK1/2 (AF1576) were obtained from R&D. Antibodies against whole form of p38MAPK (#8690), and AKT (#4691) were from Cell Signaling. p75^NTR (Santa Cruz, sc-8317) or (Abcam, ab52987). Pro-BDNF antibody was purchased from Alomone (Cat#: ANT-006). Trk-B (#610101) and sortilin (#612101) were from BD company. As loading control β-actin (Sigma-Aldrich A5441) was used. Functional assays were realized with specific antagonistic antibody to block p75^NTR (Upstate, clone ME 20.4, cat# 05-446) used at 15 ng/mL.
In some experiments, we used a broad-spectrum inhibitor of matrix metalloproteinases (MMPs) GM6001 (Calbiochem, CAS142880-36-2) (20 μM) to inhibit the cleavage of pro-BNDF in mature BDNF [21 (link)]. In all cases, GM6001 was added to culture media 30 minutes before pro-BDNF treatment.

Cultured hippocampal neurons were treated with lithium citrate for 30 min followed by a 30-min treatment with proNGF and compared with neurons treated with proNGF alone, lithium citrate alone and untreated control neurons. Cells were harvested in a buffer containing 0.6 M octylglucoside, 10% Triton X-100, 10× TNE with a phosphatase inhibitor cocktail tablet (Roche). Whole-cell lysates were precleared with protein G-Sepharose beads (Pierce) at 4°C for 60 min. The cleared lysates were incubated overnight at 4°C with α-p75^NTR (192 IgG, Millipore) followed by a 2-h incubation at 4°C with protein G-Sepharose beads. Finally, the beads were washed five times with the buffer described above, eluted by boiling in loading buffer for SDS-PAGE. Equal amounts of protein were separated by 8% PAGE, transferred to nitrocellulose membranes, and probed for sortilin (diluted 1:500, BD Sciences) and p75^NTR (diluted 1:500, Cell Signaling). All Western blot analyses were performed at least three times with samples from independent experiments.
For the in vivo experiments, hippocampi were dissected 3 d after the seizures and homogenized in RIPA buffer. Lysates were cleared with protein G-Sepharose and incubated overnight with anti-sortilin (BD Science), followed by a 2-h incubation with protein G-Sepharose beads. Samples were analyzed by Western blotting for p75^NTR (Millipore).