Sf 900 2 sfm

Drosophila S2 cells were cultured in Sf-900II SFM (Gibco) containing 50 units/ml penicillin and 50 μg/ml streptomycin at 25°C. Approximately 2×10⁵ cells were plated 24 h before transfection; 2 μg DNA were transfected into S2 cells via DDAB-mediated transfection [59 (link)]. Transfected cells were harvested after 3 days.

Spdoptera frugiperda (Sf9) cells were cultured in SF900 II SFM (Gibco) at 27 °C with orbital shaking at 120 rpm.

For expression of the recombinant BvPar j 2 and BvPar j 1 proteins 150 μl/ml of viral stock was added to 20 ml of 1 × 10⁶ cells/ml of SF9 cells in culture medium (Sf-900 II SFM, Gibco, Life technologies, Carlsbad, CA) supplemented only with gentamicin (10 μg/ml, Life technologies) but without FBS. The cells were then incubated under continuous shaking (120 rpm) for 48 hours at 27 °C. Cells and supernatant were then separated by centrifugation (1000 rpm, 10 min, 4 °C) and the supernatant was dialysed against buffer A used for purification by Ni²⁺-affinity chromatography (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM Imidazole, pH 8.0) at 4 °C. Affinity purification of recombinant proteins was achieved by using Ni-agarose (Qiagen, Hilden, Germany)⁴⁶ .
Bacterially-expressed recombinant Par j 2 was expressed and purified as described and received from Prof. Paolo Colombo^{12 (link)}.
The purity of the recombinant proteins was assessed by SDS-PAGE under non-reducing and reducing conditions with either ß-Mercaptoethanol or Tris(2-carboxyethyl)phosphine (TCEP) (see Supplementary Fig. S2). Aliquots of 2 μg of BvPar j 1, BvPar j 2 and EcPar j 2 were loaded on a 14% analytical gel^{47 (link)}. Pre-stained protein ladder (PageRulerTMPlus, Thermo Scientific) was used as a molecular weight marker to determine the apparent masses of the proteins after staining of the gels with Coomassie blue.

Spdoptera frugiperda (Sf9) cells were cultured in SF900 II SFM (Gibco) at 27 °C with orbital shaking at 120 rpm.

Full-length ORFs of BdCPR and eGFP were amplified with primers containing BamH I and Xho I (Table S1) using PrimeSTAR Max DNA Polymerase (Takara). PCR products and pFastbac HT A vector (Life Technologies) were digested with BamH I and Xho I (Thermo Scientific) for 1 h and then PCR products were inserted into vectors with T4 DNA Ligase (Promega). The vector containing the eGFP gene was used to produce a control virus. The recombinant baculovirus DNA was constructed and transfected to Sf9 cells (Life Technologies, Gibco) using the Bac-to-Bac baculovirus expression system (Life Technologies) according to the manufacturer’s instructions. Sf9 cells were suspension cultured under serum-free conditions (SF-900 II SFM, Gibco) at 27 °C. Insect cells grown to a density of 2 × 10⁶ cells mL⁻¹ were infected with recombinant baculovirus containing either BdCPR or eGFP. Baculovirus-infected cells were harvested 72 h post infection by centrifugation at 2,000 × g for 10 min and washed with PBS (100 mM, pH 7.8). Cells were resuspended in one-tenth cell culture volume of cell lysate buffer, sonicated for 1 min on ice, and centrifuged at 10,000 × g for 10 min. Supernatants were used immediately or frozen in liquid nitrogen and stored at −80 °C.

Sf9 insect cells were cultured in Sf-900 II SFM (Gibco) or HyClone CCM3 cell culture medium (Cytiva) at 27°C. The cells were splited twice a week and used until the 30th passage.