E coli max efficiency dh5α

Expression plasmids (Supplementary Table T3) were cloned using NEBuilder HiFi DNA Assembly Mix (NEB). E. coli MAX Efficiency DH5α (ThermoFisher) or C41(DE3) cells (Lucigen or Sigma) were used for plasmid propagation and protein expression, respectively. Plasmid sequences are provided in Supplementary Data D1.

Human B4GALT5 and B4GALT6 cDNAs were obtained by PCR using total RNA extracted from Huh-7 or Hep-3B cells [20 (link)] in the presence of Phusion High-fidelity Taq polymerase (ThermoFischer Scientific, Rome, Italy, Italian distributor), according to the manufacturer’s protocol. The following primer pairs were used: B4GALT5 (forward) 5′-CGCGAAGCTTGCGATCGCCATGCGCGCCCGCCGGG and (reverse) 5′-CGCGTCTAGAGTTTAAACTCAGTACTCGTTCACCTGAGCC; and B4GALT6 (forward) 5′-CGCGAAGCTTGCGATCGCCATGTCTGTGCTCAGGCGGATG and (reverse) 5′-CGCGTCTAGAGTTTAAACTTAATAGTCTTCGATTGGAGCTAACTC, both containing HindIII and XbaI restriction sites (italicized), respectively. Annealing was at 64 °C, and 30 cycles of amplification were run. The obtained fragments were purified by spin columns, digested with HindIII, repurified, digested with XbaI, purified again, and ligated to the pcDNA3 vector that was previously digested/purified with the same enzymes. E. coli Max-efficiency DH5α (ThermoFischer Scientific) was transformed by an aliquot of ligation reactions, and the obtained colonies were inoculated in liquid cultures to prepare plasmid DNA. The obtained clones were first assessed by restriction digestion and then confirmed by direct DNA sequencing.