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 (F_NCS/F_Actin) 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.