Cideb co‐precipitates with SCAP

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Cideb controls sterol‐regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites Cideb co‐precipitates with SCAP. Flag‐tagged Cideb was co‐expressed with different HA‐tagged proteins (PLIN2, Insig, SCAP, SREBP‐1, or SREBP‐2) in 293T cells and immunoprecipitated with an anti‐Flag antibody, and the co‐immunoprecipitated protein was detected by an anti‐HA antibody following SDS–PAGE. Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immunoprecipitated from primary hepatocytes under normal medium, 25‐HC depletion, or addition conditions. And the presence of Cideb in the immune complex was detected by IB. SREBP‐1 was detected as the positive control. Grp94 was detected as the negative control. Membrane topology of SCAP. The dashed line labels the portion of SCAP (a.a.416–800) that interacts with Cideb. Cideb interacts with the 416–800 portion of SCAP. Flag‐tagged Cideb was co‐expressed with the indicated HA‐tagged SCAP truncation mutants in 293T cells and immunoprecipitated with an anti‐Flag antibody. Levels of the co‐immunoprecipitated protein were detected by an anti‐HA antibody following SDS–PAGE. Sterol‐dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag‐tagged Insig‐1 or Cideb was co‐expressed with HA‐tagged SREBP‐2 and SCAP or SCAP‐Y298C in CHO‐K1 cells in sterol‐depleted medium. When indicated, 10 μg/ml 25‐HC was supplemented before cells were immunoprecipitated with an anti‐Flag antibody, and the levels of co‐immunoprecipitated proteins were detected by IB following SDS–PAGE. The dotted blue vertical line was drawn to separate the result between SCAP‐WT and SCAP‐Y298C. A “Ping‐Pong” scheme of sterol‐regulated SCAP/Insig and SCAP/Cideb interactions. SCAP switches from Insig binding to Cideb binding in the absence of sterol, while the sterol‐insensitive SCAP‐Y298C consistently interacts with Cideb. Cideb relieves sterol‐dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag‐Cideb were cultured in sterol‐deprived condition. When indicated, cells were treated with different doses of 25‐HC before subjected to IB with the indicated antibodies following SDS–PAGE. Quantification of IB data from three independent experiments. Data represent Mean ± SEM. Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP‐SCAP alone or with HA‐Cideb were cultured in sterol‐deprived conditions or treated with 1 μg/ml 25‐HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 μm (Merge) and 2 μm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi‐positive region per cell (left) and quantification of the percentage of cells containing Golgi‐localized GFP‐SCAP (right) under indicated conditions, from three independent experiments; Data represent Mean ± SEM; **P < 0.01; ***P < 0.001, by 2‐tailed Student's t‐test. Source data are available online for this figure. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 38, Issue: 8, First published: 12 March 2019, DOI: (10.15252/embj.2018100156)