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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.
