Journal: Journal of Virology
Article Title: Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum
doi: 10.1128/JVI.01904-16
Figure Lengend Snippet: RIDα regulated LD accumulation independently of Rab7 and RILP. (A and B) Representative confocal images from shNPC1-RIDα cells stained with antibodies to FLAG-RIDα (green) and HRS (red) (A) or late endosome/lysosome markers LAMP1 and LAMP2 (red) (B). (C) Postnuclear supernatants fractionated on 27% Percoll density gradients immunoblotted for RIDα and organelle-specific marker proteins (EGFR, plasma membrane; Rab5 and Rab7, early and late endosomes, respectively). (D) Equal protein aliquots immunoblotted with antibodies to Rab7 to monitor siRNA knockdown efficiency and to tubulin for protein loading control. (E) Representative confocal images from A549 cells without (top panels) or with (bottom panels) stable RIDα expression following treatment with NC or Rab7-specific siRNAs. Cells were stained with Bodipy 493/503 (green), DAPI (blue), and FLAG antibody (red). (F) RIDα immune complexes from stable cell lines expressing wild-type RIDα or RIDα (75-SS) resolved by SDS-PAGE for immunological detection with FLAG-epitope antibody. (G) Representative confocal images from stable cell lines expressing wild-type RIDα or RIDα (75-SS) stained with antibodies to β-COP (red) and FLAG-RIDα (green). (H) Representative confocal image of sterol-loaded shNPC1 cells expressing RILP-defective RIDα (75-SS) stained with FLAG-RIDα antibody (red), Bodipy 493-503 (green), and DAPI (blue). (A, G, and H) Boxed areas are magnified 2× to visualize individual and merged images to the right of each image. (A, B, E, G, and H) All size bars, 5 μm. (C and F) Representative immunoblots from two independent experiments.
Article Snippet: Antibodies and reagents used in the study are described in and , respectively. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Target Source Vendor Catalog no. Actin Rabbit Sigma (St. Louis, MO) A2066 EGFR Rabbit Fitzgerald (Concord, MA) 20R-ER004 FLAG Mouse Sigma F3165 GFP Mouse Stressgen (San Diego, CA) SAB-500 HRS Rabbit Bethyl (Montgomery, TX) A300-989A Lamin B1 Rabbit Abcam (Cambridge, MA) ab16048 LAMP1 Mouse DSHB b H4A3 LAMP2 Mouse DSHB H4B NF-κB (p65) Rabbit Cell Signaling (Beverly, MA) 8242 NF-κB (pSer536) Rabbit Cell Signaling 3033 ORP1L Mouse Santa Cruz (San Diego, CA) sc-376602 ORP1L Rabbit Abcam ab203352 ORP5 Rabbit Novus (Littleton, CO) NBP2-19683 Rab5 Mouse BD Biosciences (San Jose, CA) 610281 Rab7 Rabbit Santa Cruz sc6563 SREBP1 Mouse Novus NB600-582 TLR4 Mouse R&D Systems (Minneapolis, MN) MAB14782 TfR Mouse Zymed (South San Francisco, CA) 13-6800 Tubulin Rabbit Cell Signaling 2125 VAP-A Rabbit Sigma HPA009174 VAP-B Mouse Proteintech (Rosemont, IL) 14477-1-AP Open in a separate window a Antibodies were routinely used at a dilution of 1:1,000 to 1:2,000 for immunoblotting and at a dilution of 1:100 to 1:500 for immunostaining. b DSHB, Developmental Studies Hybridoma Bank (developed under the auspices of the Eunice Kennedy Shriver National Institute of Child Health and Human Development [NICHD] and maintained by The University of Iowa Department of Biological Sciences [Iowa City, IA]).
Techniques: Staining, Marker, Clinical Proteomics, Membrane, Knockdown, Control, Expressing, Stable Transfection, SDS Page, FLAG-tag, Western Blot
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