Henle SJ , Carlstrom LP , Cheever TR , Henley JR .

NS067311 NINDS NIH HHS , NS080322 NINDS NIH HHS , R01 NS067311 NINDS NIH HHS , F32 NS080322 NINDS NIH HHS , T32 AR007612 NIAMS NIH HHS

	FIGURE 1. Down-regulating PTEN selectively blocks MAG-induced repulsion. A, Xenopus growth cones immunostained for Akt substrate phosphorylation (pAkt sub) under control conditions, with PTEN-MO alone and PTEN-MO plus wild-type rescue (PTEN-MO + WT). Scale bar, 5 μm. B, quantification of relative Akt substrate phosphorylation immunofluorescence normalized to the control condition. C and D, example images and summary plots depict the final position of wild-type and PTEN-MO growth cones relative to the starting position (origin) after 30 min of exposure to a gradient (arrows) of MAG (C) or BDNF (D). Scale bar, 10 μm. Asterisks denote growth outside of the plot. E, cumulative distribution of growth cone turning angles in response to a gradient of medium (Control), MAG, or BDNF either alone or with PTEN-MO and PTEN-MO plus wild-type PTEN (MO + WT). F, mean turning angles and growth rates from all experiments. All data are expressed as mean ± S.E. (n = number associated with each bar; *, p < 0.05; NS, no significant difference, t test (B) and Mann-Whitney U test (F)).
	FIGURE 2. Differential function of PTEN during cAMP-dependent conversion of turning responses. A and B, summary plots depict the final position of wild-type and PTEN-MO growth cones relative to the starting position (origin) after 30 min of exposure to a gradient of MAG (A) and BDNF (B) in the presence of (Sp)-cAMPS and (Rp)-cAMPS, respectively. Scale bar, 10 μm. Asterisks denote growth outside of the plot. C, cumulative distribution of growth cone turning angles for all experiments as in A and B. D, mean turning angles and growth rates for all experiments. All data are mean ± S.E. (n = number associated with each bar; *, p < 0.05; NS, no significant difference, Mann-Whitney U test). The control group in C and D is the same as in Fig. 1, E and F.
	FIGURE 3. MAG induces PTEN-dependent depression of PIP3 levels. A–C, pseudocolor time-lapse confocal images of PHAkt-GFP standard-corrected for growth cone thickness during basal conditions (A, n = 6; see also supplemental Movie S1) and with MAG treatment (150 ng/ml; starting at time = 0) either alone (B, n = 6; see also supplemental Movies S2–S4) or after down-regulating PTEN expression (C, PTEN-MO, n = 7; see also supplemental Movie S5). Time is in minutes. White is highest fluorescence intensity. Scale bar, 5 μm. D, quantification of change in fluorescence intensity as compared with the mean intensity prior to treatment (F/F0). Treatment groups are significantly different (p = 0.0029, repeated measures two-way ANOVA).
	FIGURE 4. MAG-induced β1-integrin internalization and inhibition of clustering is PTEN-dependent. A, representative immunofluorescence images show the distribution of β1-integrin in the growth cone of wild-type and PTEN-MO neurons after experimental treatments: control (BSA), MAG (1 μg/ml; 5 min), or (Sp)-cAMPS (20 μm) + MAG. Arrowheads designate clustered β1-integrin. Scale bar, 5 μm. B and C, summary graphs show the quantification of β1-integrin surface levels (B) and β1-integrin clustering (C) for all experimental conditions. Data are the mean ± S.E. (n > 150, *, p < 0.05, one-way ANOVA with a Tukey's post hoc analysis).

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