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1.
FIGURE 4.

FIGURE 4. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

WT-ProNGF induces robust TrkA phosphorylation and neurite outgrowth. A, PC12 cells were incubated in DMEB for 1 h then treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for the indicated times, then lysed, and TrkA was immunoprecipitated. Immunoblots were performed with anti-phosphotyrosine and anti-TrkA antibodies, as indicated. B, PC12 cells were plated in serum-containing media and 24 h later, were switched to DMEB containing NGF (10 ng/ml) or WT-proNGF (50 ng/ml). Cells were maintained for 3 days and then photographed.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
2.
FIGURE 8.

FIGURE 8. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

Processing of proBDNF by metalloproteases and furin facilitates TrkB activation in cerebellar granule neurons. Rat cerebellar granule neurons were pre-incubated with dec-CMK (50 μm) or BB94 (10 μm) for 30 min, then exposed to BDNF (5 ng/ml) or WT-proBDNF (50 ng/ml) for 30 min. TrkB was immunoprecipitated with RTB, and phosphotyrosine content of TrkB in immunoprecipitates (A) and phospho-ERK levels within cell lysates (B) were determined by immunoblot. Experiments in panels A–B were repeated three times, with identical results.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
3.
FIGURE 5.

FIGURE 5. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

Inhibiting metalloproteases does not block proNGF-induced activation of TrkA signaling cascades. A, PC12 cells in DMEB were exposed to BB94 (10 μm) for 1 h, then they were treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for the times indicated, and phosphorylation of ERK and AKT was examined by immunoblot. B, PC12 cells were exposed to BB94 (10 μm) for 1 h and then treated with phorbol 12-myristate 13-acetate (PMA) (1 μm) for 90 min. p75NTR cleavage was monitored by immunoblot. Experiment in panel A was repeated three times and in panel B was repeated twice, with identical results.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
4.
FIGURE 3.

FIGURE 3. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

WT-ProNGF induces TrkA-dependent signaling cascades. PC12 cells (panels A and B) or PC12nnr5 cells (panels C and D) were incubated in DMEB for 1 h and then treated with 25 ng/ml NGF or 50 ng/ml WT-proNGF for the time points indicated. Cells were lysed in sample buffer, and phosphorylation of ERK (A and C) and AKT (B and D) were examined by immunoblot. E, PC12 cells were exposed to K252A (200 nm) for 1 h prior to addition of NGF (25 ng/ml) or WT-proNGF (50 ng/ml). Experiments were repeated three times, with identical results.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
5.
FIGURE 2.

FIGURE 2. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

AP-proNGF binds cell surface p75NTR but not TrkA. A, AP-proNGF binding assays were performed on HEK293T cells transfected with nothing (Mock), with p75NTR, or with TrkA. Values represent four wells assayed in quadruplicate. *, differences between p75NTR and mock transfection with p < 0.001. Error bars represent standard deviation. B, HEK293T cells were transfected with plasmids encoding p75NTR or TrkA, and 24 h later, cells were treated with sulfo-NHS-LC-biotin (0.5 mg/ml) for 30 min. Lysates were immunoprecipitated with streptavidin-conjugated beads and subsequently immunoblotted with p75NTR or TrkA antibodies. SA-PD, streptavidin bead pulldown. Experiments were repeated three times with identical results.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
6.
FIGURE 6.

FIGURE 6. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

Endocytosis is required for WT-proNGF treatment to induce TrkA signaling. A and B, PC12 cells in DMEB were exposed to 0.45 m sucrose for 15 min and then treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) for 10 or 30 min, as indicated. A, cells were lysed in sample buffer, and ERK phosphorylation was examined by immunoblot. B, TrkA was immunoprecipitated, and receptor phosphotyrosine levels were examined by immunoblot. C, PC12 cells in DMEB were cooled to 4 °C for 30 min and then treated with NGF (5, 10, or 50 ng/ml) or WT-proNGF (10, 50, 100 ng/ml) for 30 or 60 min, as indicated. TrkA was immunoprecipitated, and phosphotyrosine levels were examined by immunoblot. Experiments in panels A and B were repeated three times, with identical results.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
7.
FIGURE 7.

FIGURE 7. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

ProNGF cleavage is required for TrkA activation. A, PC12 cells in DMEB were pretreated with dec-CMK (50 μm) for 30 min and then exposed to NGF (25 ng/ml) or WT-proNGF (50 ng/ml). TrkA was immunoprecipitated and phosphotyrosine levels were examined by immunoblot. B, as in A with phosphorylation of ERK within cell lysates was determined by immunoblot. C, PC12 cells in DMEB were pretreated with dec-CMK (50 μm) for 30 min and then exposed to B-proNGF (25 ng/ml) for 60 min. Cells were then washed and lysed, and levels of B-proNGF and phosphorylation of ERK within cell lysates were determined by immunoblot. D, PC12 cells were treated with NGF (25 ng/ml), WT-proNGF (50 ng/ml), or proNGF123 (50 ng/ml) for the indicated times, and TrkA was immunoprecipitated and assessed for phosphotyrosine content by immunoblot. E, PC12 cells were treated with NGF (25 ng/ml) or WT-proNGF (50 ng/ml) in the absence of presence of brefeldin A (10 μg/ml). Cells were lysed in sample buffer, and levels of phospho- and total ERK and AKT as well as levels of p75NTR and TrkA were determined by immunoblot. Experiments in panels AE were repeated three times, with identical results.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.
8.
FIGURE 1.

FIGURE 1. From: Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation.

Recombinant proNGF is expressed and secreted in intact form. A, schematic representation of recombinant proNGF proteins used in this study. Position-1 refers to the cleavage site that generates fully processed NGF. AP-proNGF contains an amino-terminal AP cassette, followed by proNGF with a KR to AA mutation at the central dibasic cleavage site followed by a carboxyl-terminal Myc epitope tag. The WT-proNGF is untagged and contains the normal dibasic cleavage site at position-1. In proNGF123, the two amino acids that compose the dibasic cleavage sites at –72, –42, and –1 were mutated to alanine. B, recombinant AP-proNGF produced as described under “Experimental Procedures” was immunoprecipitated using an antibody directed against the Myc epitope tag. The immunoprecipitate was analyzed by immunoblot, together with samples of mature NGF (15 ng), WT-proNGF (25 ng), or proNGF123 (25 ng), using an antibody directed against mature NGF.

Jacqueline Boutilier, et al. J Biol Chem. 2008 May 9;283(19):12709-12716.

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