Fig. 6. Inhibiting RhoA signaling increases the amount of plasma membrane DCC in SCNs. Plasma membrane levels of the netrin 1 receptor DCC in SCN in vitro were evaluated using biotinylation (A) or immunofluorescence (B-H). Cell-surface levels were measured following 1-hour incubations with either 10 µg/ml C3-07 (C3) or 10 µM Y27632 (Y) and 5 minutes following application of 50 ng/ml netrin 1 (Net), as indicated. In the biotinylation assay (A), C3-07 caused a 50% (n=8, P=0.002) and Y27632 a 78% (n=8, P<0.001) increase in the amount of DCC in the absence of netrin 1. Netrin produced a 37% (n=8, P=0.021) increase, whereas the combination of both netrin and C3-07 or Y27632 generated a 59% (n=8, P<0.001) or 64% (n=8, P<0.001) increase, respectively. No significant change in the amount of cell-surface Tag1 was detected. (B) The ratio plasma membrane DCC immunofluorescence to total cellular DCC immunofluorescence within the growth cone increased in the presence of C3-07 by 9.9% (n=85, P=0.006), Y27632 by 8.7% (n=87, P=0.022), netrin 1 by 6.3% (n=124, P=0.048), netrin 1/C3-07 by 11.0% (n=80, P=0.002) and netrin 1/Y-27632 by 10.7% (n=90, P=0.001). Grayscale images of the plasma membrane (PM) and total DCC immunofluorescence in C-H were converted to heatmaps using the `Fire' lookup table of Image J (NIH, Bethesda, MD). ^*P<0.05, ^**P<0.01. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bar: 10 µm.