Journal: International Journal of Molecular Sciences

Article Title: ProNGF, but Not NGF, Switches from Neurotrophic to Apoptotic Activity in Response to Reductions in TrkA Receptor Levels

doi: 10.3390/ijms18030599

Figure Lengend Snippet: ProNGF causes cell death in primed PC12 cells when TrkA is knocked down, while mature NGF has no effect. ( A , B ) PC12 cells were treated with 150 nM TrkA siRNA, control siRNA, or vehicle, and primed for 18 h in 50 ng/mL 2.5S NGF. Western blot showing reduced TrkA in PC12 cells treated with TrkA siRNA, compared to control siRNA or vehicle controls. Sortilin and p75NTR levels were unchanged as a result of TrkA knockdown; ( C – F ) PC12 cells were transfected with 150 nM TrkA siRNA, control siRNA, or vehicle, primed for 18 h in 50 ng/mL 2.5S NGF, and treated for 24 h with 0.5 nM ( C ) WT-NGF, n = 11 for TrkA siRNA and control siRNA, n = 7 for vehicle ( D ) 2.5S NGF, n = 11 for TrkA siRNA, n = 12 for control siRNA, and n = 8 for vehicle ( E ) proNGF, n = 11 for TrkA siRNA, n = 12 for control siRNA, and n = 8 for vehicle; ( F ) PC12 cells were treated with 150 nM TrkA siRNA, primed for 18 h in 50 ng/mL 2.5S NGF, and treated for 24 h with 0.5 nM proNGF, WT-NGF, 2.5S NGF, or medium alone. n = 11 for proNGF, WT-NGF, and 2.5S NGF, and n = 12 for medium alone. Percentage of cell death is expressed relative to levels in medium-treated cells. For all graphs: Error bars represent SEM. *** p < 0.001. Data pooled from three independent experiments; and ( G ) Western blot of collected media showing that proNGF remained intact throughout the experiment.

Article Snippet: Western blotting was performed using affinity-purified rabbit polyclonal NGF antibody (MC-51), anti-TrkA rabbit polyclonal antibody (Chemicon, Mississauga, ON, Canada), anti-sortilin rabbit monoclonal antibody (Alomone Labs Ltd., Jerusalem, Israel), anti-p75NTR rabbit polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY, USA), anti-phosphorylated anti-extracellular signal-regulated kinase (ERK) 1/2, Thr202/Tyr204), and total ERK (Cell Signaling Technology, Inc., Mississauga, ON, Canada), anti-phosphorylated Akt (Ser473) and total Akt, or monoclonal mouse anti-β-actin antibody (clone AC-51, Sigma, Oakville, ON, Canada).

Techniques: Western Blot, Transfection

Journal: International Journal of Molecular Sciences

Article Title: ProNGF, but Not NGF, Switches from Neurotrophic to Apoptotic Activity in Response to Reductions in TrkA Receptor Levels

doi: 10.3390/ijms18030599

Figure Lengend Snippet: ProNGF is apoptotic in PC12nnr5 cells lacking TrkA. ( A ) Top panel: Representative Western blot of primed PC12nnr5 and PC12nnr5 B5 cell lysates. Each lane is derived from a separate plate. Bottom panel: quantification of A, where relative amounts of TrkA, sortilin, and p75NTR were normalized to β-actin. Similar results were obtained in a second experiment; ( B ) PC12nnr5 cells were treated for 48 h with medium alone or 0.5 nM WT-NGF, 2.5S NGF, or proNGF. White arrows show representative dead cells. Scale bars represent 5 μm; ( C ) ProNGF causes more apoptosis than medium, WT-NGF, or 2.5S NGF in PC12nnr5 cells. n = 10 for all groups. Data pooled from two independent experiments; ( D ) Re-expression of TrkA in PC12nnr5 B5 cells treated for 48 h with 0.5 nM proNGF, WT-NGF, or medium alone. n = 14 for proNGF and WT-NGF and n = 15 for 2.5S and medium alone. Data pooled from three independent experiments. Percentage of cell death is expressed relative to levels in medium-treated cells. Error bars represent SEM. *** p < 0.005; and ( E ) Western blot of collected media showing that proNGF remained intact throughout the experiment.

Article Snippet: Western blotting was performed using affinity-purified rabbit polyclonal NGF antibody (MC-51), anti-TrkA rabbit polyclonal antibody (Chemicon, Mississauga, ON, Canada), anti-sortilin rabbit monoclonal antibody (Alomone Labs Ltd., Jerusalem, Israel), anti-p75NTR rabbit polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY, USA), anti-phosphorylated anti-extracellular signal-regulated kinase (ERK) 1/2, Thr202/Tyr204), and total ERK (Cell Signaling Technology, Inc., Mississauga, ON, Canada), anti-phosphorylated Akt (Ser473) and total Akt, or monoclonal mouse anti-β-actin antibody (clone AC-51, Sigma, Oakville, ON, Canada).

Techniques: Western Blot, Derivative Assay, Expressing