Research Paper Volume 14, Issue 18 pp 7568—7586

Novel TRKB agonists activate TRKB and downstream ERK and AKT signaling to protect Aβ-GFP SH-SY5Y cells against Aβ toxicity


Figure 6. Activation of ERK, AKT and CREB pathways downstream to TRKB in Aβ-GFP SH-SY5Y cells. (A) Experimental flow chart. On day 1, cells were plated with retinoic acid (RA, 10 μM) added to the culture medium. On day 2, LMDS-1 or -2 (5 μM) was added to the cells for 8 h, followed by inducing Aβ-GFP expression with doxycycline (Dox, 5 μg/ml). Kinase inhibitors U0126 or wortmannin (10 μM) were added to the cells on day 6. On day 8, ERK, AKT, TRKB, CREB, BDNF, BCL2 and BAX levels were measured. (B) p-ERK (T202/Y204), ERK, p-AKT (S473), AKT, (C) p-TRKB (Y516 and Y817), TRKB, p-CREB (S133), CREB, BDNF (31/13 kDa), BCL2 and BAX levels analysed by immunoblot using GAPDH as a loading control (n = 3). To normalize, protein expression level in untreated cells was set at 100%. P values: comparisons between induced vs. uninduced cells (#P < 0.05, ##P < 0.01), compound-treated vs. untreated cells (*P < 0.05, **P < 0.01, ***P < 0.001), or kinase inhibitor-treated vs. untreated cells (&P < 0.05, &&&P < 0.001). (one-way ANOVA with a post hoc Tukey test).