Figure 3.
Inhibition of TRPC5 expression by siRNA in NG108-15 cells. Four designated TRPC5 siRNA sequences (Table 2) were transfected into undifferentiated NG108-15 cells; control transfection used TRPC6 siRNA sequences. A, RT-PCR assay 48 h after transfection. siRNA 1, 2, and 4 sequences knocked down TRPC5 only, whereas siRNA 3 reduced both TRPC5 and TRPC1. TRPC6 was not affected by this siRNA treatment. The designation (−) indicates vehicle transfection. B, Immunoblot 48 h after transfection with TRPC5 siRNA 2, showing inhibition of TRPC5 protein synthesis. Cort., Positive control with protein extract from cortical tissue of a 7-d-old rat pup. C, IC images in cells transfected with TRPC5 siRNA 2. After 48 h (a) and 72 h (b) treatment, cells were stained with anti-TRPC5 Ab and HRP-linked secondary Ab. TRPC5 expression was inhibited 48 h after transfection and partially recovered 72 h after transfection. Cells treated with vehicle alone (c) expressed TRPC5 in both cytosol and plasma membrane (arrows). D, NG108-15 cells were treated with TRPC6 siRNA for 48 h and subjected to RT-PCR, showing selective loss of TRPC6 mRNA (a). b, c, TRPC6 siRNA-treated cells were strained with TRPC5 Ab (b) and TRPC6 Ab (c), which verified TRPC6 knock-down. d, Vehicle-transfected cells were stained with TRPC6 Ab.