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. 2018 Oct 1;217(10):3747–3765. doi: 10.1083/jcb.201802057

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© 2018 Risher et al.

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Figure 6. — TSP-induced synapse formation requires Rho GTPase Rac1. (A) Rat cortical neuron purification and TSP2 treatment timeline. (B) Rat cortical neurons transfected with a pX601 vector containing saCas9 and sgRNA identified by HA expression. The pX601-only control (top) did not include an sgRNA sequence. Neurons were treated with TSP2-containing or deficient growth media. Colocalized pre- (VGluT1) and postsynaptic (PSD95) puncta reveal sites of excitatory synapses (yellow arrowheads). (C) Excitatory synapse density on HA⁺ neurons (n = 30 cells per condition; two independent experiments). (D) GFP⁺ rat cortical neuron dendrites. After transfection with an shRNA-expressing vector (with a scrambled shRNA sequence [shScr] as a control), neurons were treated with TSP2-containing or -deficient growth media. Colocalized pre (Bassoon) and postsynaptic (Homer) puncta reveal sites of excitatory synapses (yellow arrowheads). (E) Excitatory synapse density on GFP⁺ neurons (n = 30 cells per condition; two independent experiments). Error bars represent SEM. (F and G) Same scheme as D and E except using shRNA against the Rho GEFs Kalirin-7, Tiam1, and β-Pix. One-way ANOVA with Tukey’s multiple comparisons post hoc test. Bars, 5 µm. *, P < 0.05; ***, P < 0.0001.