Fig. 2.

P75 increases sensitivity of TrkA cells. (A) Cells were seeded in serum-free medium, and NGF (100 ng/ml) was added after overnight incubation. Cells were continuously monitored by the RT-CES system. Cell Index, which reflects cell number in the plates, was recorded every 5–30 min. The vertical black line (in 2A) indicates the time point when NGF was added. Transient changes (within minutes) in CI immediately following neurotrophin addition represent cytoskeletal modulation that may occur following receptor phosphorylation [28]. Points are the averaged normalized CI of six replicate wells, (bars=SD). The difference in normalized CI between SY5Y-TrkA and SY5Y-TrkA/p75 treated with NGF is significant. (*p<0.01). The difference of normalized CI between TrkA and TrkA/p75 without added NGF is not significant. Graph represents a single experiment (repeated at least three times with similar results). (B) MTT assay performed at end point of RT-CES experiment, average OD of triplicate wells measured (error bars, ±SD). The difference in OD between SY5Y-TrkA and SY5Y-TrkA/p75 treated with NGF is significant (*p<0.01). (C) Flow cytometry analysis shows effect of NGF on cell cycle after 18 h treatment. The difference of G1 and S+G2/M phase percent changes by NGF between TrkA and TrkA/p75 cells is significant (p<0.05). ). Graph represents mean of five experiments (bars=SD). (D) Phosphorylation of TrkA and AKT (Ser473) was examined by immunoblotting after treatment with indicated NGF concentrations. The blots were stripped and re-probed with anti-Trk and anti-AKT antibodies.