Hsc70 associates with a DCC multiprotein signaling complex and supports DCC cell surface localization downstream of netrin-1. (A) Isolated E17.5 rat cortices were stimulated with netrin-1 for the indicated times. DCC was IP and the level of Trio, FAK, and Hsc70 coIP with DCC was assessed by immunoblotting. TCL, total cell lysates. (B) Densitometric analysis of Hsc70 coIP with DCC. Error bars indicate SEM (n = 4; NS, P > 0.05; *, P < 0.05; one-way ANOVA, Dunnett’s multiple comparisons test). (C) Cortical neurons were dissociated and treated with netrin-1 at DIV2, followed by surface DCC isolation. Subsequent IP of lysates depleted of surface DCC were performed and the level of Hsc70 coIP with DCC was assessed by immunoblotting. (bottom) Densitometric analysis of Hsc70 coIP with either surface or intracellular DCC. Error bars indicate SEM (n = 4; NS, P > 0.05; *, P < 0.05; unpaired student’s t test). (D) Isolated E17.5 rat cortical neurons were electroporated with the indicated Hsc70 constructs. At DIV1 the neurons were treated with netrin-1 for 15 min before fixation. Surface DCC was assessed by staining with an extracellular DCC antibody under nonpermeabilizing conditions and subsequent fluorescence microscopy. Arrowhead denotes enchrichment and # denotes no enrichment. Bar, 50 µm. (E) The mean pixel intensity ratios of surface DCC at the growth cones relative to axons were calculated using Metamorph software (>54 neurons assessed per condition, from four independent experiments). Error bars indicate the SEM (**, P < 0.01; unpaired student’s t test). (F) 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 before lysis. Protein extracts were resolved by SDS-PAGE and the levels of active ERK1/2 and FAK were assessed by immunoblotting. (G and H) Densitometric analysis of pERK1/2 and pFAK (Y861) over total proteins from F. Error bars indicate SEM (n = 4; *, P < 0.05; unpaired student’s t test).