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Fig. 3. NF-
B-dependent gene expression in newborn nodose neurons is not
affected by BDNF. (A) Photomicrographs of representative fields of neurons
co-transfected with the GFP NF-B reporter plasmid, RFP plasmid and
either the super-repressor IB-
plasmid (right panels) or
corresponding control plasmid (left panels) and cultured with 20 ng/ml BDNF
for 12 hours. Transfected neurons and their dendritic morphology are outlined
by the RFP (shown with a white filter) and can be seen in the upper panels.
The effect of super-repressor IB- on neuronal morphology, seen
in the previous figures, is also evident in these images. GFP fluorescence in
the same fields (lower panels) shows the marked reduction caused by
super-repressor IB-. (B) Quantification of the level of
NF-B-driven GFP fluorescence in super-repressor
IB--transfected and control-transfected neurons after the
neurons have been incubated for 12 hours with and without 20 ng/ml BDNF.
Fluorescence measurements were made from 40 to 60 neurons in each experimental
condition, and the data are expressed as a percentage of the mean fluorescence
of the No factor/Control vector-transfected group (mean and standard error are
shown). Statistical comparisons shown are with respect to the control
transfected neurons, **P<0.001. (C) Timecourse of
NF-B-driven GFP fluorescence after BDNF stimulation. Neurons were
co-transfected with the GFP NF-B reporter plasmid and the RFP plasmid,
and were cultured without factors for 8 hours. Neurons were then imaged
immediately before and at 0.5, 1, 2 and 3 hours after the addition of BDNF to
the medium (20 ng/ml). An untreated group of neurons (No Factor) was imaged at
the same times. The fluorescence of each neuron was quantified and expressed
as a percentage of the initial (0 hour time point) measurement for each group.
Scale bars: 50 µm.
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