Anti insig 1

25HC, 27HC, 24SHC, 24,25EC, 7βHC, and ISRIB were procured from Sigma–Aldrich. 7KC was procured from Santa Cruz Biotechnology. 7αHC was procured from Abcam. Thapsigargin was procured from Wako. The following antibodies were procured from commercial sources: anti-ATF4, anti-eIF2α, antiphosphorylated eIF2α, and anti-PERK (code: 11815, 9722, 9721, and 3192) from Cell Signaling Technology; anti-SREBP2 and anti-INSIG1 (code: sc-13552 and sc-390504) from Santa Cruz Biotechnology; anti-β-actin, anti-FLAG, and anti-c-Myc (code: A5441, F1804, and C3956) from Sigma–Aldrich; anti-GFP (code: ab6556) from Abcam; and peroxidase-conjugated antimouse IgG and peroxidase-conjugated anti-rabbit IgG (code: 715-035-151 and 111-035-144) from Jackson ImmunoResearch. Anti-INSIG2 rabbit polyclonal antibody was produced by Cosmo Bio with an epitope to amino acids 215 to 225 of human INSIG2. Anti-HMGCR mouse monoclonal antibody (clone A9) and anti-Chinese hamster SREBP2 mouse monoclonal antibody (clone 7D4) were generous gifts from Dr Ta-Yuan Chang (Geisel School of Medicine). All other chemicals of analytical grade were obtained from Sigma–Aldrich, Wako, or nacalai tesque.

Immunoblotting was performed as previously described39 . The following antibodies were used in this study: anti-CREBH, anti-Insig-1 and anti-Insig-2 (Santa Cruz, USA); anti-SREBP-1 and anti-SREBP-2 (Novus, USA); anti-Flag (Cell signaling, USA). All antibodies were used at a final concentration of 0.1–1 μg/ml. After incubation with the appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (1:5000 dilution; GE Healthcare UK), proteins were visualized by enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (Amersham Biosciences, Pittsburgh, PA, USA).