Express five serum free medium

KIR2DL5-Ig was generated in an inducible secreted serum-free Drosophila expression system as described previously (48 (link), 66 (link)). Briefly, the coding region of the extracellular domain without signal peptide of KIR2DL5 was fused to a human IgG1 Fc tag in a pMT/BiP vector. Construct was cotransfected with a blasticidin-resistant plasmid into Drosophila Schneider 2 (S2) cells by the calcium phosphate transfection kit (Invitrogen). The stably transfected S2 cells were selected and expanded in Schneider’s Drosophila Medium (Gibco) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL blasticidin (Gold Biotechnology). The S2 cells were induced to secrete fusion proteins in Express Five serum-free medium (Life Technologies) in the presence of 0.75 mM CuSO₄. Proteins were purified using Protein G resin (GenScript) columns.

The genes encoding A. aegypti salivary protein were cloned without signal sequences into a pMT/BiP/V5-His A vector (Cat# V4130-20, Invitrogen) for expression in Drosophila S2 cells. For immunoprecipitation assays, the human LRPPRC gene, LRPPRC truncations, PTCD1 gene, and Beclin-1 gene were cloned into a pcDNA3.1/Myc-His C vector for expression in 293T cells. LRPPRC and LRPPRC-T3 were purified from 293F cells using a Cobalt-His column. Beclin-1 was cloned into a pGEX-6P-2 vector and purified from E. coli using glutathione sepharose. The cloning primers are shown in Supplementary Table 4. For AaVA-1 expression, the stable S2 cells were cultured in S2 Schneider’s medium in a 175 cm² flask and then transferred into spinner flasks containing Express Five serum-free medium (Cat# 10486-025, Gibco) for protein expression. The cells were further cultured for 3 days and induced with 500 μM copper sulfate for another 4 days. The supernatant was centrifuged, filtrated, and then concentrated for AaVA-1 purification by a TALON Purification Kit (Cat# 635515, Clontech).

Drosophila S2 cells (CVCL_Z232) originally from William Chia’s laboratory (with a non-authenticated identity but have been used in the laboratory for the past 10 years) were cultured in Express Five serum-free medium (Gibco) supplemented with 2 mM Glutamine (Thermo Fisher Scientific). S2 cell culture used in this study is free of contamination of Mycoplasma, due to the absence of small speckles of DAPI staining outside of the cell nucleus. For transient expression of proteins, S2 cells were transfected using Effectene Transfection Reagent (QIAGEN) according to manufacturer’s protocol. S2 cells were harvested 48 hr after transfection and were homogenized using lysis buffer (25 mM Tris pH8/27.5 mM NaCl/20 mM KCl/25 mM sucrose/10 mM EDTA/10 Mm EGTA/1 mM DTT/10% (v/v) glycerol/0.5% Nonidet P40) with Complete Proteases inhibitors (Roche) for 30 min at 4°C. Cell lysates were subjected to SDS-PAGE and western blotting.