Fab fragments were prepared using immobilized papain

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macia Biotech). FAb fragments were prepared using immobilized papain (Pierce, Rockford, IL). For treatment with FAbs, sphingolipids, and sphingolipid synthesis inhibitors, neurotrophins were used at 30 ng/ml. For NGF competition, BDNF was used at 3 ng/ml. Detection of phosphorylated Trk and mitogen-activated protein kinase. Subplate neurons cultured for 24 hr were treated with 100 ng/ml neuro- trophin for 40 min for detection of phosphorylated mitogen-activated protein kinase (MAPK) or for 5 min for detection of phosphorylated Trk. To block Trk signaling, neurons were incubated with 200 n M K252a for 1 hr before addition of neurotrophin. Equal numbers of neurons were extracted with 30 m l of extraction buffer (20 m M Tris, pH 7.5, 250 m M NaCl, 0.5% Triton X-100, 3 m M EGTA, 3 m M EDTA, 20 m M b -glycerophosphate, 0.1 m M NaVO 4 , 50 m M NaF, 5 m g/ml chymostatin, 5 m g/ml leupeptin, 5 m g/ml antipain and 5 m g/ml pepstatin, and 1 m M PMSF) and scraped off the culture dish. Extracts were spun at 10,000 rpm for 10 min. Equal amounts of supernatant were analyzed by SDS- PAGE and standard immunoblotting techniques. Nitrocellulose blots (Schleicher & Schuell, Dassel, Germany) were blocked with Superblotto (Pierce) and incubated with a 1:5000 dilution of anti-active MAPK (catalog #V6671; Promega) or with a 1:750 dilution of anti-phospho- TrkA (Tyr490) [(catalog #9141S; New England Biolabs, Beverly, MA) this antibody cross-reacts with phosphorylated TrkB and TrkC]. The secondary antibody was 1:10,000 HRP-conjugated donkey anti-rabbit (Jackson ImmunoResearch). Blots were visualized by enhanced chemi- luminescence (NEN). RESULTS Subplate neurons express p75NTR and Trks Subplate-restricted expression of p75NTR, as well as coexpres- sion of Trks, was revealed by in situ hybridization using Trk and p75NTR-specific [ 35 S]-riboprobes on sections of E17 rat cerebral cortex (Fig. 1). Subplate neurons were labeled with a pulse of BrdU at E12, when they are undergoing their final round of cell division. Because subplate and marginal zone neurons are the only cortical neurons that become postmitotic on E12 in rat, they are the only cells subsequently heavily labeled with BrdU (Fig. 1) (Bayer and Altman, 1990). Note that, because subplate neurons in rat are generated over a period of several days (E12–E15), only a fraction of them are heavily labeled by a single injection of BrdU. p75NTR expression is restricted to the meninges and the sub- plate zone and colocalizes with the region of heavily BrdU- labeled subplate neurons. On the other hand, TrkB and TrkC are expressed throughout the cortical plate, the subplate, and the ventricular zone. In contrast, TrkA is not detected in cortex at this age. Thus, whereas TrkB and TrkC are expressed by many post- mitotic neurons, embryonic expression of p75NTR is restricted to those neurons known to later undergo programmed cell death.
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