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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: FEBS Lett. 2020 Feb 6;594(9):1389–1402. doi: 10.1002/1873-3468.13740

Figure 2. The EPAC-specific cAMP analogue 8-CPT activates Rap1 and overcomes inhibition by MAG when used for priming.

Figure 2.

(A) Western blots of P5-6 CGN treated with 8-CPT (0.5 mM) for 20 minutes, or with MAG-Fc (20 μg/ml) for 20 minutes prior to the addition of 8-CPT (n=3). Lysates were used for Rap1 activation assays detecting GTP-bound Rap1, and total Rap1 was assessed using input samples. (B) Representative images of P5-6 CGN that were incubated overnight with 1 mM dbcAMP, 0.25, 0.5 or 1 mM 8-CPT, and transferred to monolayers of either MAG-expressing CHO cells or control CHO cells (scale bar=10 μm). Graph depicts average length of the longest neurite per neuron ±SEM for approximately 150-200 neurons per treatment (3 independent experiments, ***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparisons test). (C). Quantification of neurite outgrowth for P5-6 CGN that were treated with 1 mM dbcAMP, 0.5 or 1 mM 8-CPT, and plated directly on monolayers of MAG-expressing CHO cells or control CHO cells. Graph depicts average length of the longest neurite per neuron ±SEM for approximately 150-200 neurons per treatment (3 independent experiments, **p < 0.01, one-way ANOVA with Bonferroni’s multiple comparisons test).