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

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Figure 4. — The neurotoxic mutant PrP(AV3) forms heterodimers with WTPrP under physiological conditions. A, schematic representation of the constructs used. The serine variants of WTPrP and PrP(AV3) were modified with an HA (WTPrP-HA and PrP(AV3)-HA) and a V5 tag (WTPrP-V5 and PrP(AV3)-V5). The tags were inserted after amino acid 35. B, HeLa cells were transiently transfected with the indicated constructs. Cells were lysed, and HA-tagged PrP was immunoprecipitated under nonreducing conditions with HA-agarose beads. The immunopellet was dissolved in Laemmli sample buffer containing β-mercaptoethanol and analyzed by Western blotting (WB) using an anti-V5 antibody. Western blot analysis of the inputs is shown below. Asterisk, signal corresponds to primary antibody used in the immunoprecipitation (IP).