FIGURE 1.

Expression of Tm6sf2 and generation of Tm6sf2^−/− mice. A, total RNA was extracted from the indicated tissues of WT female mice (n = 3, age = 14 weeks) after a 4-h fast and subjected to quantitative real-time PCR as described under “Experimental Procedures.” The mean ± S.E. (error bars) levels of TM6SF2 transcript in each tissue are expressed relative to the expression level in the ileum, which was arbitrarily set to 1. B, regulation of TM6SF2 expression in response to fasting and refeeding. Mice were entrained to a synchronized feeding regimen for 3 days and then killed after a 24-h fast (Fasted) or after 18 h of fasting and 6 h of refeeding (Refed) (4 male mice, age = 8 weeks). Jejunal and liver proteins (60 μg/well) were size-fractionated by 10% SDS-PAGE, and immunoblotting analysis was performed using antibodies against TM6SF2 and calnexin. C (left), liver proteins (60 μg/well) from the experiment described in B were size-fractionated on an SDS-10% polyacrylamide gel. Fatty acid synthase (FAS) and adipose TG lipase (ATGL) and were used as positive controls for fasting and refeeding, and calnexin (CNX) was used as a loading control in this experiment. C (right), immunoblotting signals were quantified using a LI-COR Odyssey Fc imager. D, Tm6sf2^−/− mice were generated as described under “Experimental Procedures.” Genotyping was performed by PCR using oligonucleotides (arrows) to amplify a 470-bp (WT) or 400-bp (KO) fragment from genomic DNA. E (left), RNA was isolated from livers of male WT and KO mice (n = 3 male mice/group, 14 weeks old), and TM6SF2 expression was determined by quantitative real-time PCR as described under “Experimental Procedures.” The level of TM6SF2 transcript in WT mice was arbitrarily set to 1. E (right), immunoblotting analysis of hepatic TM6SF2 in 7-week-old female WT and KO mice. Liver lysates and membranes were prepared as described under “Experimental Procedures.” Aliquots of each fraction (50 μg) were size-fractionated by SDS-PAGE, and immunoblotting was performed using a rabbit anti-mouse TM6SF2 polyclonal antibody (1:1000) as described under “Experimental Procedures.” Calnexin served as a loading control for the experiment. *, nonspecific band. All experiments were repeated at least once, and the results were similar. Ct, cycle threshold. Values are means ± S.E. **, p < 0.01. AU, arbitrary units.