Cells were transfected with scrambled siRNA or NCDase siRNA for about 36 hours and then cells were collected, lysed and then subjected to RT-PCR, Western and NCDase activities assays as described under “Materials and Methods”. (A) Real time RT-PCR analysis of NCDase mRNA level. (B)Western blot analysis of endogenous NCDase expression in H. end. cells. Equal loading was verified by using anti-β-actin. (C) TLC analysis of NCDase activity in NCDase siRNA treated cells. (D) NCDase activity was quantified by densitometry. The values are expressed as the mean ±SD (n=3). Results are representative of three independent experiments. SCR, scrambled siRNA; NCD, NCD siRNAi; FA, fatty acid product; NBD CER, NBD C-12 ceramide substrate. Statistical significance was calculated with respect to scrambled control (*, p<0.05)