Extended Data Fig. 1: In vitro ubiquitination assay of HMGCR.

a, Quantification of the proteins in Fig. 1a. The signals of HMGCR, FDFT1, LSS and DHCR24 were normalized to that of GAPDH. The amount of each protein in fasted state was defined as 1. b, Relative amounts of mRNAs in the livers of the mice in Fig. 1a measured by qPCR. c, Immunoblot analysis of HMGCR, FDFT1, LSS and DHCR24 in the livers of mice fasted (F) for 12 h, or fasted for 12 h and then refed (R) with a high-carbohydrate/low-fat diet for 3 h, 6 h, or 12 h (n = 3 per group) respectively. d, Quantification of proteins in (c). e, The mRNA levels of indicated genes in mouse liver (n = 4 per group) treated as in (c). f, Schematic representation of the experimental design for in vitro ubiquitination assays. Membrane fractions were prepared from the sterol-depleted CHO-7 cells to provide un-ubiquitinated HMGCR and E3 complex. The liver cytosols were prepared from mice under fasted or refed conditions. The membrane fractions were incubated with E1, UBE2G2, ATP, FLAG-Ubiquitin, the indicated cytosol and 25-hydroxycholesterol (25-HC) at 37 °C for 30 min. Samples were solubilized and HMGCR was immunoprecipitated with polyclonal anti-HMGCR antibodies. Immunoblotting was carried out with monoclonal anti-FLAG or monoclonal anti-HMGCR antibodies. g, In vitro ubiquitination of HMGCR as described in (f). Experiments were performed as indicated three times with similar results. All values are presented as mean ± SEM. Data were analysed by unpaired two-tailed Student’s t-test (a, b), or one-way ANOVA with Tukey’s multiple comparisons test (d, e).

Source data