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FGF18 has a lower stringency for HS sulfation than other FGFs as it recognizes both 2-O-sulfated and 6-O-sulfated HS oligosaccharides, with a preference for 2-O-sulfation (Ashikari-Hada et al., 2004).
It is possible that the higher affinity provided by arginine residues in a HS-binding interface could explain why FGF18 has a lower stringency for HS sulfation than other FGFs.
Alongside with these high-stringent requirements, data analysis with lower stringency was performed.
Moreover, in both genomes, BLASTER gave a higher genome coverage than PALS (Table S1), as expected given the lower stringency of its parameters (see Methods).
The higher positive rate (53%) of GO sharing suggests lower stringency of GO term matches when making an inference of PPIs.
We did not reanalyse the data using lower stringency but instead aim in future experiments to extend the current study to additional brain regions and samples.
Finally, we demonstrated that the dimerization between the pathogenic variants also occurred with lower stringency as indicated by the relative activity of the lacZ reporter gene.
The lower stringency for the presence of distal N-glycans at s3 and s4 is consistent with the conservation of these sites during evolution.
Finally, we performed a location analysis of the M1 and M11 matches in the human genome and found that 80% of the matches were at ZNF genes; for M1: 569 of 724 top matches and 968 of 1198 lower stringency matches; for M11: 148 of 190 top matches and 571 of 703 lower stringency matches.
The relatively lower sensitivity (66%) and specificity (65%) of GO sharing may be explained by lower stringency of GO term matches as described above, suggesting that it is not a strong indicator of PPI.
This lower validation rate was expected for two reasons: (i) the lower stringency could enhance the noise; (ii) we only used one amplicon per locus, thus probably missing nearby p63 binding sites.
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