FIG 7.

Ad2-induced cholesterol homeostasis regulated TLR4/NF-κB signaling. (A) Equal protein aliquots from shNPC1 cells with wild-type RIDα or palmitoylation-defective RIDα mutant (C67S) resolved under reducing (+DTT) or nonreducing (−DTT) conditions to detect RIDα monomers and dimers. (B) Stable cell lines listed in the figure analyzed for endogenous NPC1 and ORP1L expression by immunoblotting. (C) Representative confocal images of RIDα (C67S)-expressing shNPC1 cells stained with FLAG-RIDα antibody (red), Bodipy 493/503 (green), and DAPI (blue) (left) or LAMP1 antibody (red) and filipin (blue) (right), following standard LDL-loading protocol. The boxed area in the right panel is enlarged 2× to visualize individual channels. All size bars, 5 μm. (D) Equal protein aliquots from untreated (−) hepatocyte-derived cells or following treatment with LPS immunoblotted with activation-specific phospho-NF-κB/p65 (Ser536) antibody and reprobed for total NF-κB/p65 to verify equal protein loading. (E) Equal protein aliquots from parent hepatocytes infected with WT-Ad2 or an Ad2 mutant encoding RIDα (C67S) immunoblotted for RIDα at 6 or 12 h postinfection (p.i.). (F) Equal protein aliquots from parent hepatocytes that were mock treated or infected with wild-type or Ad2 mutants listed in the figure for 20 h immunoblotted with receptor-specific antibodies (EGFR, TLR4, and transferrin receptor [TfR]). (G) Equal protein aliquots from mock-treated cells or cells infected with Ads listed in the figure for 24 h and then left untreated (−) or stimulated with LPS for 30 or 60 min, immunoblotted with activation-specific phospho-NF-κB/p65 (Ser536) antibody, and reprobed for total NF-κB/p65 to verify equal protein loading. (D to G) Representative immunoblots from 2 independent experiments.