Figure 3

Crosslinked heterodimers of prion protein. Representative SDS-PAGE gel pictures of the photo-crosslinked reaction mixtures of various mPrP (“UV irradiated”, + UV) (a) and their control, non-irradiated (“Non-irradiated controls”, Dark) (b) counterparts. In case of all reaction-mixtures presented, pBpa is present in the untagged mPrP at the position 127 (Y127pBpa). The partner protein in each case is a prion-mCherry fusion protein with or without possessing a G126V mutation in the sequence of the prion. The reaction mixtures irradiated at 365 nm (+) and non-irradiated (−) for 2 h, are in presence of either 0.06% or 2% SDS in order to promote dimerization or to assess the background non-specific association of the proteins, respectively. The expected positions of the two kinds of monomers (the untagged mPrP and/or its mutant variants at 23 kDa, and of the mPrP-mCherry fusion protein and/or its valine mutant variant at 52 kDa) and of their heterodimer (75 kDa) is indicated on the figure. Lanes 1 (“M”) contain the same molecular weight marker loaded. The two bands appearing at 28 kDa and 46 kDa on the gels are cleavage products, originating from the partial hydrolysis of the main-chain acylimine linkage of the chromophore of mCherry, a phenomenon known to occur for DsRed-like chromophores upon sample-treatment, such as boiling in presence of SDS and dithiothreitol for SDS-PAGE analysis⁷². The vertical black lines delineate the position of cropping, where lanes unrelated to the figure were removed from the gel pictures (panel b). The images of the full-length gels are provided in Supplementary Fig. S2).