Hsc70 is required for netrin-1–mediated cortical axon outgrowth and Rac1 activation. (A) Dissociated E17.5 rat cortical neurons were electroporated with control siRNA or Hsc70 siRNA with a GFP reporter construct or the indicated GFP constructs. At DIV1, neurons were stimulated with netrin-1 or glutamate for 24 h. Bar, 50 µm. (B and C) The mean axon lengths of GFP⁺ neurons from A were calculated manually using Metamorph software (>50 neurons assessed per condition, from at least three independent experiments). Error bars indicate the SEM (****, P < 0.0001; one-way ANOVA, Fisher’s least significant difference post-test). (D) E17.5 rat cortical neurons were depleted of endogenous Trio and Hsc70 by siRNA and reexpressed both with siRNA-resistant Hsc70 and Trio. (E and F) The mean axon lengths of GFP⁺ neurons were calculated as in B (>70 neurons assessed per condition, from at least three independent experiments). Error bars indicate the SEM (*, P < 0.05; unpaired t test). (G) HEK293 cells were transfected with pRK5-DCC with empty vector (EV), GFP-Hsc70, or GFP-Hsc70^D10N and stimulated with netrin-1 for 5 min. GTP-loaded Rac1 was pulled down from protein lysates by GST-CRIB. GTP-bound Rac1 (top), total Rac1, and the indicated proteins were detected by immunoblotting. TCL, total cell lysates. (H) Densitometric ratio of GTP-bound Rac1 to total Rac1 normalized to DCC. Error bars indicate the SEM (n = 6; *, P < 0.05; one-way ANOVA, Bonferroni’s multiple comparisons test).