FIGURE 2.

p120RasGAP interacts with DCC in netrin-1-induced embryonic cortical neurons. E18 rat cortical neurons were stimulated with netrin-1 (500 ng/ml) for the indicated times after being cultured for 2 DIV. A, p120RasGAP was immunoprecipitated (IP) from cell lysates with anti-p120RasGAP antibodies or mouse IgG as a control. Immunoprecipitated proteins and total cell lysates (TCL) were resolved by SDS-PAGE and immunoblotted (IB) with antibodies against the indicated proteins. B, quantitative densitometry (mean ± S.E.) of DCC co-immunoprecipitated with p120RasGAP is represented as the -fold change relative to 0 min of netrin-1 stimulation for at least three independent experiments (unpaired Student's t test; ***, p < 0.005; ns, not significant). C, E18 embryonic rat cortical neurons (2 DIV) were incubated with 500 ng/ml netrin-1 or left unstimulated for 10 min, immunostained with antibodies against DCC and p120RasGAP, and imaged by confocal microscopy. Arrows indicate cell bodies, and arrowheads indicate growth cones. The gray dashed outlines represent growth cones. Scale bars = 50 μm (whole cells) and 20 μm (growth cones). D, the correlation between DCC and p120RasGAP fluorescence intensities in C was measured with Metamorph software using Pearson's correlation coefficient (mean ± S.E.) in whole cells (wc) and growth cones (gc) in three independent experiments (number of neurons = 65, 51, 54, and 40 from left to right; unpaired Student's t test; ns, not significant; *, p = 0.028). n, netrin-1; −, unstimulated.