Generation of Mammalian N1 Mutants

Mammalian expression plasmids encoding mouse LFNG (pAPtag2-LFNG), full-length mouse N1 (pcDNA1-N1), and O-fucose site mutants of mouse N1 (pcDNA1-N1-EGF6V, pcDNA1-N1-EGF26V, pcDNA1-N1-EGF27V) were described previously (4 (link)). The RFP plasmid was obtained from Addgene (#12520), the TP1-1 luciferase reporter construct was kind gift from Dr Georg Bornkamm, and the gWIZ β-galactosidase construct was from Gene Therapy Systems. The O-fucose site mutant in N1 EGF16 (pcDNA1-N1-EGF16V) was generated by PCR-directed mutagenesis with primers (Table S9) and CloneAmp HiFi (TAKARA). All mutants were confirmed by sequencing.