Figure 8
From: The G127V variant of the prion protein interferes with dimer formation in vitro but not in cellulo
V127PrP forms disulfide bond-linked heterodimers with G127PrP. (a) Scheme of the experimental strategy to specifically detect G127/V127 heterodimers with disulfide bond stabilization. A cysteine was inserted as S132C mutation. HA-tagged G127PrP was first immunoprecipitated under non-reducing conditions using anti-HA agarose beads. In a second step Western blot analysis under non-reducing conditions with anti-V5 antibody revealed co-purified V5-tagged V127PrP in the immunopellet. The tags were inserted after aa 35 (b) HeLa cells were transiently transfected with either HA-tagged G127PrP, or V5-tagged V127PrP or both. Cells were lysed and HA-tagged WTPrP immunoprecipitated under non-reducing conditions with HA-agarose beads. The immunopellet was analyzed by Western blotting under non-reducing conditions using an anti-V5 antibody. Western blot analysis of the input controls is shown below. White arrowhead: monomer, black arrowhead: dimer. Asterisk: signal corresponds to primary antibody used in the IP.
