Hsc70 facilitates Trio-dependent Rac1 activation and cortical axon outgrowth in a chaperone-dependent manner. (A) Schematic of Trio domain structure. (B) Trio constructs were transfected into HEK293 cells as indicated. GFP-Trio proteins were IP from cell lysates and Hsc70 coIP with Trio was detected by immunoblotting. TCL, total cell lysates. (C) Densitometric analysis of Hsc70 coIP with GFP-Trio from B. Error bars indicate the SEM (n = 3; NS, P > 0.05; **, P < 0.001; ***, P < 0.0001; one-way ANOVA, Bonferroni’s multiple comparisons test). (D) HEK293 cells were transfected with the indicated constructs and with increasing amounts of GFP-Hsc70 plasmid. GTP-loaded Rac1 was pulled down from protein lysates by GST-CRIB. GTP-bound Rac1, total Rac1, and the indicated proteins were detected by immunoblotting. (E) Densitometric ratio of GTP-bound Rac1/total Rac1 normalized to the vector control. Error bars indicate the SEM (n = 4; NS, P > 0.05; *, P < 0.05; ***, P < 0.0001; one-way ANOVA, Bonferroni’s multiple comparisons test). (F) Dissociated E17.5 rat cortical neurons were electroporated with the indicated constructs and a GFP reporter. Bar, 50 µm. (G) The mean axon lengths of GFP⁺ neurons were calculated manually with Metamorph software (>54 axons per condition from three independent experiments). Error bars indicate the SEM (NS, P > 0.05; **, P < 0.001; ***, P < 0.0001; one-way ANOVA, Bonferroni’s multiple comparisons test). (H) Representative protein expression of electroporated constructs in E17.5 cortical neurons from F and G. WT, wild-type Hsc70; D10N, Hsc70^D10N.