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Fig. 2. 27-mer dsRNA knockdown of NCS-1 in L.
stagnalis. (A,B) Western blot analysis of
ganglia protein extracts isolated 2 days after the snails were injected with
either NCS-1 dsRNA (NCS dsRNA), control dsRNA (Ctrl dsRNA)
or water (Ctrl), showing that NCS-1 dsRNA reduced the protein level
of NCS-1. (A) Representative semiquantitative immunoblots of NCS-1 and
β-actin expression in the central ganglia with indicated pretreatments.
(B) A summary of normalized intensity of NCS-1 to β-actin ratio from
dsRNA-treated groups compared with that from the control group. The solid line
indicates the NCS-1 to β-actin ratio of the control group. NCS-1
dsRNA treatment (2 µl of 20 µM) resulted in an average 35.7±5.1%
(n=4) reduction in the normalized relative protein expression of
NCS-1 to β-actin, whereas the control dsRNA treatment did not affect the
relative expression level of NCS-1 (2.7±2.5% reduction).
(C,D) Confocal fluorescence images obtained from double
immunocytochemical staining of β-actin and NCS-1, under different dsRNA
treatments (5 nM) for 24 hours. (C) Representative β-actin and NCS-1
immunofluorescence images of PeA neurons in culture for 24 hours. (D) Summary
of the NCS-1 levels from various regions of the culture PeA neurons. Relative
fluorescence intensities of NCS to β-actin ratio
(FNCS/FActin) for the control dsRNA group: soma
1.48±0.08; neurite 1.51±0.12; branch point 1.44±0.05; and
growth cone 1.60±0.12. The relative ratio for NCS dsRNA group:
soma 0.84±0.06; neurite 0.94±0.13; branch point
0.97±0.08; growth cone 1.10±0.16. The data are presented as
mean±s.e.m. and are obtained from a total of 10 control and 15
NCS-1-specific dsRNA-treated cells. Asterisk indicates significant
difference (P<0.05) between the control and NCS-1
dsRNA-treated conditions.
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