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. 2017 Feb 28;91(6):e01904-16. doi: 10.1128/JVI.01904-16

FIG 5.

FIG 5

RIDα stabilized ORP1L-VAP protein contacts. (A) Left panel: CE quantification in LDL-loaded shNPC1 cells or in shNPC1-RIDα cells following treatment with NC or 3′-UTR ORP1L-specific siRNAs using an Amplex Red assay. Right panel: ORP1L-depleted cells reconstituted with the siRNA-resistant GFP-tagged ORP1L constructs described in the Fig. 3 legend. Both panels: values were normalized to total cellular protein (means ± s.e.m.; n = 3; **, P < 0.005). (B) Representative confocal images from ORP1L-depleted cells reconstituted with siRNA-resistant GFP-ORP1L constructs (green) prior to sterol depletion/LDL loading. Cells were stained with LipidTOX reagent (red) to detect LDs. (C) GST or GST-RIDα-CT immobilized on glutathione beads incubated with recombinant GFP-ORP1L protein followed by recombinant Myc-tagged VAP-B protein. Reactions were also carried out in reverse order with Myc-tagged VAP-B added to GST fusion protein immobilized on glutathione beads first followed by GFP-ORP1L. Bound proteins were resolved by SDS-PAGE for immunological detection of recombinant proteins with native antibodies or GST to verify equal protein loading. (D) Left panels: cells listed in the figure expressing GFP-tagged WT-ORP1L. Right panels: shNPC1-RIDα cells expressing GFP-tagged ORP1L mutant constructs listed in figure. Both panels: GFP-tagged ORP1L proteins were affinity purified on anti-GFP-Sepharose beads, and immobilized proteins were resolved by SDS-PAGE for immunological detection of VAP-A isoforms I and II. Blots were reprobed for GFP to verify GFP-ORP1L affinity purification. (C and D) Immunoblots are representative of results of 2 independent experiments. (E) Representative confocal images from shNPC1-RIDα cells stained with antibodies to FLAG-RIDα (green) and ORP1L (left panel) or VAP (right panel) (both in red). (B and E) Boxed areas enlarged 2× to highlight LDs (B) or individual, merged, and masked (green and red pixels above the mean red or green intensity) images (E). Arrows indicate examples of colocalized signals. All size bars, 5 μm.