Fig. 4.

Effect of dietary OTC supplementation on nSMase-2 protein in mouse livers. Young and aged C57Bl6 mice were placed on either a 0.5% OTC or a SAA def. control diet for 4 weeks. nSMase-2 (A, B) and nSMase-1 (C, D) protein levels in whole liver lysates were determined by Western blot using specific rabbit polyclonal antibodies against each protein. Cyclophilin-A was used to control for equal loading and for normalization. Mean values ± SD (n = 3–4 animals per group) of the ratio between the respective nSMase protein and the loading control are shown. ASMase activity (E) was determined in whole liver homogenates as described in the Materials and Methods. nSMase-2 specific activity (F) was calculated as the total nSMase activity measured for each individual mouse divided by the nSMase-2 protein amount quantified by Western blotting. Data are shown as mean values ± SD (n = 3–4 animals per group). Statistical analyses were done according to two-way ANOVA and Bonferroni posttest analyses. The statistical significance of the main effect of diet and aging is shown by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001). No statistically significant interaction effects were present.