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. 2010 Dec 15;30(50):16766–16776. doi: 10.1523/JNEUROSCI.1362-10.2010

Figure 4.

Figure 4.

GSK3β mediates JIP3 restriction of axon branching. A, Lysates of granule neurons nucleofected with the U6/JIP3i or control U6 RNAi plasmid were immunoblotted with the JIP3 and GSK3 antibodies. 14-3-3β served as the loading control. Knockdown of JIP3 reduced the levels of GSK3β but not GSK3α in neurons. B, RT-PCR was performed to quantify the relative mRNA abundance of GSK3β in granule neurons nucleofected with the U6/JIP3i or control U6 RNAi plasmid. Relative mRNA abundance was calculated for the U6/JIP3i condition relative to the U6 condition, normalized by GAPDH. Knockdown of JIP3 significantly decreased the relative levels of GSK3β mRNA (t test, p < 0.05). C, Lysates of granule neurons nucleofected with the U6/GSK3βi1, U6/GSK3βi2, or control U6 RNAi plasmid were immunoblotted with the GSK3 and HSP60 antibodies. GSK3β RNAi induced knockdown of GSK3β but not GSK3α in neurons. D, Granule neurons transfected with the U6/GSK3βi1, U6/GSK3βi2, or control U6 RNAi plasmid together with the GFP expression plasmid were analyzed as in Figure 1B. Scale bars, 50 μm. GSK3β knockdown triggered the formation of exuberant axon branches. E, Quantification of axon branching of granule neurons treated as in D. For either hairpin, the number of axonal tips and the number of axonal self-contact points per hundred micrometers were both significantly increased in GSK3β knockdown neurons compared with control U6-transfected neurons (ANOVA followed by Fisher's PLSD test, p < 0.001 for all). A total of 257 neurons were measured. F, Granule neurons transfected with the U6/JIP3i, U6/GSK3βi2, or both RNAi plasmids or the control U6 RNAi plasmid together with the GFP expression plasmid were analyzed as in Figure 1B. Knockdown of either JIP3 or GSK3β alone significantly increased the number of axonal tips and the number of axonal self-contact points per hundred micrometers (ANOVA followed by Fisher's PLSD test, p < 0.01 or p < 0.001 as specified by asterisks). Knockdown of JIP3 and GSK3β together did not increase the number of axonal tips or self-contact points compared with knockdown of JIP3 or GSK3β alone. A total of 273 neurons were measured.

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