To assay the effect of these mutations on splicing we opted to introduce the mutated intron sequences at their endogenous locus, instead of within the CUP1 gene as was previously done [ 8, 9].
Moreover, the effect of these mutations was investigated for the full-length protein.
The effect of these mutations on membrane binding was determined by a quantitative phospholipid ELISA assay and compared to wild-type α-Syn and to the Parkinson's disease-causing mutations, A30P, E46K and A53T.
To verify the effect of these mutations on the transport activity of hENT1, a structural model for hENT1 was generated (Fig. S4).
We examined the effect of these mutations on the expression of the reporter gene chloramphenicol acetyltransferase, which was expressed from a truncated thymidine kinase promoter fused to the renin regulatory region.
The effect of these mutations on nuclear localization was easily discernible.
The effect of these mutations on the 21OH activity is unknown.
The effect of these mutations on biotinyl-lysine cleavage was not determined.
However, an effect of these mutations on alternate mGluR1 signalling pathways has not been excluded [33].
We assessed the effect of these mutations on antiviral activity and DNA editing, both individually and in combination.
This must occur later in the ISGylation process, since no effect of these mutations can be observed in the absence of the N89D mutation.
