Figure 1. Accumulation of HMGCR protein in livers of Ubiad1^Ki/Ki mice with mixed C57BL/6 × 129 genetic background.

(A) Amino acid sequence and predicted topology of mouse UBIAD1 protein. Asparagine-100 (N100), which corresponds to the most frequently mutated amino acid residue in SCD, is enlarged, shaded in red and indicated by an arrow. (B) Male WT, Ubiad1^WT/Ki, and Ubiad1^Ki/Ki littermates (8–9 weeks of age, eight mice/group) were fed an ad libitum chow diet prior to sacrifice. Livers of the mice were harvested and subjected to subcellular fractionation as described in ‘Materials and methods.’ Aliquots of resulting membrane (Memb.) and nuclear extract (N.E.) fractions (80–160 µg of total protein/lane) for each group were pooled and subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against endogenous HMGCR, SREBP-1, SREBP-2, UBIAD1, Insig-1, Insig-2, calnexin, and LSD-1. Although shown in a separate panel, LSD-1 serves as a loading control for the nuclear SREBP immunoblots. The amount of hepatic HMGCR protein in Ubiad1^Ki/Ki mice was determined by quantifying the band corresponding to HMGCR using ImageJ software.

Figure 1—figure supplement 1. Relative amounts of hepatic mRNAs encoding components of the Scap-SREBP pathway and lipid analysis in WT and Ubiad1^Ki/Ki mice.

(A) Total RNA isolated from livers of mice used in Figure 1B (8 mice/group) was separately isolated. Equal amounts of RNA from the individual mice were subjected to quantitative real-time RT-PCR using primers against the indicated gene; cyclophilin mRNA was used as an invariant control. Each value represents the amount of mRNA relative to that in WT mice, which is arbitrarily defined as 1. Bars, mean ± S.E. (error bars) of data from eight mice. (B) The amount of cholesterol, triglycerides, and non-esterified fatty acids (NEFA) in livers and plasma from WT or Ubiad1 knockin mice used in Figure 1B was determined by a colorimetric assay as described in ‘Materials and methods.’ Error bars, S.E. The p value was calculated using Student’s t test: *, p ≤ 0.05. Hmgcs, HMG coenzyme A synthase; Fpps, farnesyl pyrophosphate synthase; Sqs, squalene synthase; Acs, acetyl coenzyme A synthetase; Acc1, acetyl coenzyme A carboxylase-1; Fas, fatty acid synthase; Scd-1, stearoyl coenzyme A desaturase-1; Gpat, glycerol-3-phosphate acyltransferase; Abcg5 and Abcg8, ATP-binding cassette subfamily G member 5 and 8, respectively; Ggpps, geranylgeranyl pyrophosphate synthase.