FIG 1.

RIDα compensated for loss of NPC1 function following an acute infection. (A) A549 cells infected with Ad2 (wild type or RIDα-null) metabolically labeled from 1 to 3 h postinfection and harvested 8 h postinfection to recover E1A and RIDα immune complexes for detection by fluorography. IP, immunoprecipitation. (B) Equal protein aliquots from mock-treated or Ad-infected cells immunoblotted (IB) with antibodies to NPC1 (top) and actin (bottom) for protein loading control. (C) Representative confocal images from A549 cells mock treated or Ad infected for 18 h and stained with antibodies to LAMP1 (red) and NPC1 (green) and filipin (blue) to visualize free cholesterol. Boxed areas are magnified 2× to visualize individual channels beneath each image. Nu, nucleus. (D) A549 cells mock treated or Ad infected and stained with Bodipy 493/503 (green) and DAPI (blue) to detect LDs and nuclei, respectively. (E) Equal protein aliquots from NPC1-positive hepatocytes (shControl) and NPC1 knockdown hepatocytes without or with stable expression of FLAG-tagged RIDα (shNPC1 and shNPC1-RIDα, respectively) immunoblotted with antibodies to NPC1 (top) and FLAG (middle) and actin for protein loading control (bottom). (F) Mock-treated or Ad-infected shNPC1 cells stained with Bodipy 493/503 (green) and DAPI (blue). (C, D, and F) All size bars, 5 μm. (G) Equal protein aliquots of cytosol and nuclear fractions from sterol-depleted (−) and LDL-loaded (+) cells listed in the figure probed with SREBP-1 antibody to detect precursor (P) and nuclear (N) forms of the molecule. Nuclear fractions were reprobed for lamin B1 to verify equal protein loading. Representative immunoblots from 2 independent experiments are shown.