Pfuse higg1 fc1

Human and mouse NPP1 (Human: NCBI accession NP_006199; Mouse: NCBI accession NP_03839) modified to express soluble, recombinant protein as described previously26 (link) were fused to IgG₁ by subcloning into pFUSE-h IgG₁-Fc1 or pFUSE-m IgG₁-Fc1 plasmids (InvivoGen, San Diego, CA), respectively. The final human protein sequence is listed in Supplementary Fig. 1.

RNA was extracted from 5 × 10⁶ to 10 × 10⁶ hybridoma cells using TRIzol reagent (Invitrogen, Gaithersburg, MD). Heavy (H)- and light (L)-chain cDNAs containing the gene encoding the antibody-binding (Fab) region of 3G9 were amplified and sequenced, as previously reported^{79 (link)}. Then, primer sets were designed to clone the Fab regions of H and L chains into pFUSE-hIgG1-Fc1 and pFUSE2-CLIg-hL2, respectively (InvivoGen, San Diego, CA). Gene cloning was performed following the manufacturer’s instructions (primer information is available upon request).
Then, three kinds of mutation [L234A/L235A(LALA), D265A, and N297A] that abolished Fc-Fc receptor interaction were introduced into the Fc region using a site-directed mutagenesis kit, following the manufacturer’s protocol (TOYOBO, Osaka, Japan)^{47 (link)}.
The plasmids containing H- or L-chain genes (50 μg each) were transduced to 1 × 10⁸ 293F cells using 293fectin reagent (ThermoFisher Scientific, Waltham, MA). After incubating the cells at 37 °C for 4 days, the culture fluids were harvested and purified by protein G (GE Healthcare, Chicago, IL). The concentration of purified IgG was calculated by measuring the absorbance at 280 nm. Purified IgG was then used in neuralization tests, ADE assays, and animal experiments (see below).

The Trp-containing dendrimer MADAL385 was synthesized as described previously [38 (link)]. Vapendavir and pirodavir were kindly provided by Aviafen Therapeutics and A. Muigg, respectively. Suramin sodium salt and heparin sodium were purchased from Sigma-Aldrich. All compounds were dissolved in DMSO and stored at 4°C. The plasmid pEV-A71 (Nagoya-VP231) was generously provided by Dr. Arita (National Institute of Infectious Disease, Japan) and was modified to carry the viral genome of EV-A71 BrCr (pEV-A71-BrCr) by classic cloning. For the construction of EV-A71BrCr-mCherry, the genome of EV-A71 BrCr was inserted to a pShuttle-BAC vector. In the spacer region between the 5'UTR and VP4, mCherry gene was inserted and flanked by 2A^pro cutting sites. The plasmids pLX304-PSGL-1 was purchased from DNASU, and vector pFUSE-hIgG1-Fc1 was from InvivoGen. PSGL-1 extracellular region was cloned into the expression vector pFUSE-hIgG1-Fc1 to create recombinant protein with human IgG Fc-tag at the C-terminal domain (pPSGL1-hFc). The EV-A71 VP1 purified MaxPab rabbit polyclonal antibody was obtained from Abnova. Goat anti-Human IgG Fc cross-absorbed secondary antibody, HRP was purchased from Thermo Scientific. Heparin Sepharose CL-6B and Dynabeads Protein G were supplied by Pharmacia and Thermo Scientific, respectively.