To confirm that expression of Spn4A.RRLL(s) in Huh-7.5.1 cells inhibits endogenous SKI-1/S1P-mediated cleavage of SREBP molecules, we examined nuclear SREBP-1 protein levels in cells infected with adenovirus-expressed Spn4A variants. As a positive control, we also treated cells with the selective, reversible, and competitive small-molecule inhibitor of SKI-1/S1P: PF-429242 [64, 65] . This compound was recently synthesized and characterized both in vitro and in vivo for its anti-lipidemic properties including efficient inhibition of nuclear SREBP accumulation. As previously described [64, 66] , the calpain inhibitor, alpha-N-acetyl-Leu-Leu-Nle-CHO (ALLN), was employed to facilitate the detection and accumulation of the Nterminal fragment of SREBP-1 in the nucleus. An anti-fibrillarin antibody was used to positively identify the nuclear fractions ( Figure 3A ) [67] . As expected, Western blotting of nuclear extracts from cells treated with 10 mM of PF-429242 (PF-429242 + ALLN), using an antibody against the N-terminal fragment of SREBP-1, revealed a complete block of SREBP-1 accumulation in the nucleus ( Figure 3A ). Nuclear extracts from cells infected with Ad-Spn4A.RRLL(s) (RRLL(s) + ALLN) also exhibited a dramatic decrease in nuclear SREBP-1 accumulation when compared to Ad-Empty (control + ALLN) and Ad-Spn4A.RRLL(r) (RRLL(r) + ALLN)-infected cells. These results confirm that expression of recombinant Spn4A.RRLL(s) in the secretory pathway of Huh-7.5.1 cells inhibits SREBP-1 processing by SKI-1/S1P.