Insig1 isoform abundances in response to sterol abundance. (A) Cells were depleted of sterols for 14 h (O/N) in media containing lipoprotein-deficient serum, lovastatin, and low levels of mevalonate. At time zero, sterols and mevalonate were added back. (Lower panel) Western blot to visualize Insig1 and HMGCR protein under conditions of sterol starvation and during addback, in the presence and absence of the proteasome inhibitor, MG132. The band marked by an asterisk reacts nonspecifically with the Insig1 antibody and serves as an internal loading control. (B) Stabilities of Insig1 mRNA isoforms during depletion and following add-back of sterol/mevalonate. Huh7 cells were depleted of sterols for 14 h. At time zero, sterols and sterol intermediates were added and RNA was harvested at the indicated times. Insig1 mRNA isoform abundances were measured by Northern blot analyses. Insig1 isoform RNA abundance was normalized to actin mRNA at each time point (lower panel). Autoradiographs were quantitated using ImageQuant (GE Healthcare). RNA abundance at time zero was set to 100%. Error bars represent standard error of the mean. (C) Polysomal distribution of Insig1 mRNA isoforms. Cells were either depleted of sterols, or depleted and re-fed for four hours. Lysates were prepared and analyzed by sucrose-gradient analysis, as described in Materials and Methods. The relative abundances of the three Insig1 mRNA isoforms in each fraction under sterol-depleted and sterol-fed conditions were measured in Northern blots. (Lower panels) Representative Northern blot displaying Insig1 isoform RNAs in each gradient fraction. Autoradiographs were quantitated using ImageQuant (GE Healthcare). The data are representative of at least three independent replicates.
