Figure 3.

Both synaptic activity and muscle contraction modulates TrkB phosphorylation at tyr816. Panels (A–E) show Western blot bands and their quantification. (A) pTrkB.FL in presynaptic stimulation treatment, contraction treatment and presynaptic stimulation with contraction treatment at 1 Hz stimulation for 30 min. Presynaptic stimulation has been simplified as Stimulation. Each column has been compared to its respective control (see Table 1). pTrkB did not show any change after 30 min of increased activity. (B) pTrkB.FL at 1, 10 and 30 min under presynaptic stimulation and presynaptic stimulation with contraction treatments. Each time-point has been compared to its previous time-point. Presynaptic stimulation significantly increased pTrkB.FL at 1 min, an effect maintained after 10 min but declined significantly near baseline by 30 min of electrical stimulation. In the presence of muscle contraction, pTrkB.FL levels decreased after 1 min and increased back to baseline levels at 30 min of treatment. (C) Phosphatase inhibition prevented the decrease of pTrkB.FL under presynaptic stimulation with contraction at 1 and 10 min. (D) Effect of the sequestering antibody 47/TrkB (10 μg/ml) on pTrkB.FL under presynaptic stimulation and presynaptic stimulation with contraction treatments (1 Hz stimulation for 30 min). After both treatments, pTrkB.FL levels decreased. (E) Effect of exogenous BDNF (10 μM) on pTrkB.FL following 30 min under presynaptic stimulation and presynaptic stimulation with contraction treatments (1 Hz stimulation for 30 min). pTrkB.FL did not change under these conditions. Specific phosphorylation was determined as the ratio of phosphorylated protein to total protein content. Data are mean percentage ± SEM, *p < 0.05. Abbreviations: pTrkB.FL, phosphorylated TrkB.FL.