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. 2018 Apr 10;293(21):8020–8031. doi: 10.1074/jbc.RA117.000990

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Figure 3. — The neurotoxic mutant PrP(AV3) forms disulfide bond–linked heterodimers with WTPrP. A, schematic representation of the mutants used. The cysteine variants of WTPrP and PrP(AV3) were modified with an HA (WTPrP-HA) or a V5 tag (PrP(AV3)-V5). The tags were inserted after amino acid 35. B, scheme of the experimental strategy. To specifically detect WT/AV3 heterodimers, HA-tagged WTPrP was first immunoprecipitated under nonreducing conditions using anti-HA-agarose beads. To detect copurified V5-tagged PrP(AV3), the immunopellet was then analyzed by Western blotting under nonreducing conditions using an anti-V5 antibody. C, N2a cells were transiently transfected with either HA-tagged WTPrP, V5-tagged PrP(AV3), or both. Cells were lysed, and WTPrP was immunoprecipitated under nonreducing conditions with HA-agarose beads. The immunopellet was analyzed by Western blotting (WB) under nonreducing conditions using an anti-V5 antibody. Western blot analysis of the inputs is shown below. White arrowhead, monomer; black arrowhead; dimer. Asterisk, signal corresponds to primary antibody used in the immunoprecipitation (IP).