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Files in this Data Supplement:
Fig. S1. Specific P2X7 antagonists potentiate axon growth and branching in the nanomolar range. (A) Hippocampal neurons were grown in the presence of BBG at a concentration of 100 or 500 nM. Neurons were fixed after 3 days in culture and they were stained with an anti-tubulin antibody (green) and phalloidin-Alexa-Fluor-594 (red). Axon length and ramifications were quantified using the Neuron J program. The graphs represent the mean and s.e.m. of three independent experiments (n=100 neurons/experiment). (B) Neurons grown for 3 days in the presence of the P2X7 antagonist A-438079. Graphs represent the mean and s.e.m. from three independent experiments (n=100 neurons/experiment). **P<0.01, ***P<0.001, ****P<0.0001, paired t-test. Scale bar: 50 µm.
Fig. S2. (A) Control hippocampal neurons and neurons exposed to BBG were stained with a pCAMKII antibody. Note the stronger expression of pCAMKII in the soma and dendrites than in the axon, and the weaker staining of pCAMKII at axon terminals in BBG treated neurons. Scale bar: 50 µm. (B) Quantification of total pCAMKII in relation to the total CAMKII in western blots of control neurons and those exposed to BBG for 30 and 60 minutes. The graph represents the mean and s.e.m. from three independent experiments.
Fig. S3. BBG antagonizes and reverses the negative effect of ATP on axon growth. Hippocampal neurons were grown for 3 days in the presence or absence of ATP (1 mM), with or without BBG (5 mM). The neurons were fixed and stained with an anti-tubulin antibody, and the axon length and number of ramifications was quantified. Graphs represent the mean and s.e.m. of three independent experiments (n=100 neurons/experiment, *P<0.05, **P<0.01, ****P<0.0001, paired t-test). Scale bar: 50 µm.
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