The molecular chaperone Hsc70 associates with Trio in the developing cerebral cortex. (A) Trio was IP from lysates of isolated E17.5 rat cortices with anti-Trio antibodies or rabbit IgG as a control. IP proteins and total cell lysates (TCL) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Trio isoforms: FL, full length; D, Trio-D; A, Trio-A. (B) Isolated E17.5 rat cortices were stimulated with netrin-1 for the indicated times. Trio isoforms were IP and Hsc70 coIP with Trio was assessed by immunoblotting. Netrin-1 stimulation of DCC-induced signaling pathways was assessed by evaluating FAK phosphorylation (Y861) in total cell lysates (TCL). (C) Densitometric analysis of Hsc70 coIP with Trio from B. Error bars indicate the SEM (n = 5; NS, P > 0.05; **, P < 0.001; ***, P < 0.0001; one-way ANOVA, Bonferroni’s multiple comparisons test). (D and G) Dissociated E17.5 rat cortical neurons were stimulated with netrin-1 for 5 and 15 min before fixation. Endogenous Trio and Hsc70 localization were assessed by indirect immunofluorescence and confocal microscopy. Bars, 15 µm. (E and F) The mean Pearson’s correlation coefficient between green (Hsc70) and red (Trio) channels within the growth cone (D and E) or axon shaft (F and G) was calculated with Metamorph software. Error bars indicate the SEM (>48 neurons were assessed per condition, from three independent experiments; NS, P > 0.05; **, P < 0.001; ***, P < 0.0001; one-way ANOVA, Bonferroni’s multiple comparisons test).