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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Neuropharmacology. 2017 Feb 1;117:93–105. doi: 10.1016/j.neuropharm.2017.01.034

Figure 4. Stimulation of muscarinic receptors in SH-SY5Y cells and NMDA receptors in N2a cells causes TGase-dependent Rac1 activation.

Figure 4

A. Carbachol treatment increased Rac1 activity in SH-SY5Y cells. SH-SY5Y cells were treated with vehicle or 1mM carbachol, a muscarinic receptor agonist, for 5min and 10min, and Rac1 activity was detected. One-way ANOVA shows a significant difference among groups [F(2,6)=13.85, p<0.01]. Newman-Keuls multiple comparison test indicates * p<0.01, n=3.

B. Carbachol-stimulated Rac1 transamidation is TGase dependent. Pretreatment with cystamine prevents carbachol-stimulated Rac1 transamidation. Two-way ANOVA analysis shows a significant effect of cystamine pretreatment, F(1,8)=90.978, p<0.001], significant effect of carbachol stimulation [F(1,8)=31.28, p<0.001], and a significant interaction between cystamine and carbachol treatment on Rac1 transamidation [F(1,8)=59.003, p<0.001]. Newman-Keuls multiple comparison test indicates * p< 0.001, n=3 compared with non-stimulated in vehicle pretreated cells; # p<0.001, n=3 compared with carbachol-stimulated controls in vehicle-pretreated cells.

C. Carbachol-stimulated Rac1 activation is TGase dependent. Cystamine pretreatment inhibited carbachol-stimulated activity of Rac1. Two-way ANOVA: main effects of cystamine pretreatment [F(1,8) = 9.405, p<0.05], main effects of carbachol stimulation [F(1,8)=3.155, p=0.114], and interaction between cystamine pretreatment and carbachol treatment [F(1,8)=5.134, p=0.053]. Newman-Keuls multiple comparison test indicates * p< 0.05, n=3 compared with non-stimulated in vehicle pretreated cells; # p<0.01, n=3 compared with carbachol-stimulated controls in vehicle-pretreated cells.

D. NMDA stimulates Rac1 activation in N2A cells. N2a cells were treated with 50μM NMDA or 100μM AMPA or vehicle for 5 minutes. NMDA increased Rac1 activation and pretreatment with the TGase inhibitor cystamine prevented this increase. Stimulation of AMPA receptors did not increase Rac1 activity at either 5 (data not shown) or 15 minutes post-stimulation. One-way ANOVA: F(3,8) = 6.234, p= 0.017, Newman-Keuls multiple comparison test indicates that NMDA-treated cells were significantly different from vehicle-treated cells (shown by *) and # indicates cells treated with both cystamine and NMDA were significantly different cells treated with NMDA alone at p < 0.05.