Figure 2.

Caltubin siRNA treatment significantly reduces expression of both caltubin transcript and protein in vivo and in vitro. Relative expression levels of ganglial mRNA (A, B) or protein (C, D) of caltubin against β-actin were measured 24 h following injection of 2 μl of solution containing saline, control siRNA, or caltubin siRNA (LCa1, LCa3, or LCa10) into the snail central ganglia. A, C, Representative gel image and Western blot. B, D, Quantification of caltubin mRNA and protein levels. Caltubin siRNA treatment, but not control siRNA treatment, significantly reduced the expression of caltubin mRNA (41 ± 1.3%; n = 3) and protein (54 ± 1.6%; n = 3). E, Representative confocal images of caltubin and β-actin double-labeled PeA cells cultured in the presence of caltubin or control siRNA. F, Summary of caltubin to β-actin ratios in soma, neurites, and growths of cultured PeA cells treated with control or caltubin siRNA, showing a significant reduction in caltubin levels specifically in caltubin siRNA-treated cells. Soma: Control siRNA, n = 11; caltubin siRNA, n = 10. Neurites: Control siRNA, n = 12; caltubin siRNA, n = 10. Growth cones: Control siRNA, n = 12; caltubin siRNA, n = 11. G, H, Time course of caltubin siRNA treatment and caltubin protein expression. G, Representative confocal images of immunocytochemical staining of PeA cells at various time points following caltubin siRNA (LCa 3) treatment. Scale bar, 20 μm. H, A significant reduction in relative caltubin protein levels was already observed at 3 h [1.19 ± 0.13 (n = 9; p < 0.05)] and 6 h [0.81 ± 0.06 (n = 10; p < 0.05)] following siRNA treatment, compared with 0 h [1.92 ± 0.94 (n = 11)]. All the data are presented as mean ± SEM (n). *p < 0.05.