Vivaspin

Protein was obtained as previously described in [35 (link)]. In brief, HEK293T cells were transfected with the plasmids encoding NS1 and grown for two days in serum-free medium. Then, cell culture fluids were collected, cell debris was removed by centrifugation and samples were concentrated by VivaSpin (Sartorius, Mw 100 kDa). Concentrated samples were subjected to an FPLC column of Sephadex 200. High-molecular weight protein fractions (90 kDa–300 kDa) were collected and analyzed by Western blot with anti-NS1 antibodies (clone 4C4, Biosan, Novosibirsk, Russia) (Supplementary Figure S1A,B). Fractions with maximum amounts of NS1 were pooled, concentrated with VivaSpin (Sartorius, Mw 5 kDa) and analyzed in gel followed by Coomassie blue staining. Cell culture fluids of cells transfected with pVax were processed the same as NS1-containing samples and were used as a control.

The virulence factor production and real virulence of P. aeruginosa were measured using Tenebrio molitor larvae by injecting the culture supernatants or live bacterial cells, respectively, as described previously (19 (link), 20 (link)). The aliquots were taken from the P. aeruginosa culture at 6 h and 24 h, and cells were removed by centrifugation at 4°C. The supernatants were filtered through a 0.2 μm filter (GVS Abluo Syringe Filter) and concentrated by a 10-kDa cutoff Centricon (Vivaspin, Sartorius). Five μL of the concentrated supernatants, insect saline (130 mM NaCl, 5 mM KCl, and 1 mM CaCl₂) or LB were injected into T. molitor larvae using a syringe. Insect saline (IS) and LB were both used as controls. For the live cell injection, the cultured cells were diluted to 1.12 × 10⁵ CFU/mL in insect saline and 5.6 × 10² CFU (5 μL) was injected into each T. molitor larva by syringe. The larvae were incubated in petri dishes at 25°C and the surviving larvae were counted every 24 h. Survival rate was presented as a percentage (%).

The virulence factor production and real virulence of P. aeruginosa were measured using Tenebrio molitor larvae by injecting the culture supernatants or live bacterial cells, respectively, as described previously (19 (link), 20 (link)). The aliquots were taken from the P. aeruginosa culture at 6 h and 24 h, and cells were removed by centrifugation at 4°C. The supernatants were filtered through a 0.2 μm filter (GVS Abluo Syringe Filter) and concentrated by a 10-kDa cutoff Centricon (Vivaspin, Sartorius). Five μL of the concentrated supernatants, insect saline (130 mM NaCl, 5 mM KCl, and 1 mM CaCl₂) or LB were injected into T. molitor larvae using a syringe. Insect saline (IS) and LB were both used as controls. For the live cell injection, the cultured cells were diluted to 1.12 × 10⁵ CFU/mL in insect saline and 5.6 × 10² CFU (5 μL) was injected into each T. molitor larva by syringe. The larvae were incubated in petri dishes at 25°C and the surviving larvae were counted every 24 h. Survival rate was presented as a percentage (%).

NNV (SgNag05 isolate, RGNNV genotype, serotype C)^{17 (link),18 (link)} was cultured using SSN-1 cells^{5 (link)} maintained at 25 °C with Leibovitz’s L-15 medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin–streptomycin solution (Gibco) (L-15₁₀ medium). NNV particles were purified by methods described previously^{19 (link)–22 (link)}. Cultured NNV suspension was centrifuged at 12,000 × g for 20 min at 4 °C. The resultant supernatant was dialyzed using a Biotech cellulose ester (CE) membrane tube with a molecular weight cut off (MWCO) of 10⁶ (Spectrum Laboratories). After anion-exchange chromatography with a Hi-trap Q column (GE Healthcare), NNV particles were eluted with 700 mM NaCl. The resultant NNV suspension was desalted and concentrated by centrifugal ultrafiltration using a membrane with MWCO of 10⁵ (Vivaspin, Sartorius).

The variants of PTPN4 named F620G, Q621G, Y52G, I623G and ΔFQYI were obtained by using the Quik-Change Mutagenesis kit (Stratagene) according to the manufacturer’s instructions, and the substitutions were confirmed by DNA sequencing. Template DNA used for PTPN4 mutants was the PDZ-PTP_WT construct cloned in a pET15b expression plasmid. The PTP construct was obtained by using the Pwo polymerase (Roche) according to the manufacturer’s instructions, and the insertion was confirmed by DNA sequencing. Template DNA used for the PTP construct was a synthetic construct comprising the PTP domain cloned in a pgex6p1 expression plasmid. Expression and purification of linker-PTP and PTP construct were performed as described previously^{13 (link)}. Bidomain PDZ-PTP variants were expressed and purified as previously described without TEV cleavage. Briefly, clarified supernatants were loaded on Ni²⁺ column (HiTrap Chelating HP, GE), washed and eluted with an imidazole gradient from 40 mM to 1 M. The eluted fractions containing the protein were pooled and loaded on a gel filtration column (Hiload Superdex 75 pg, GE). Proteins were concentrated using centrifugal filter devices (Vivaspin, Sartorius). Protein concentration was estimated from its absorbance at 280 nm. The peptide Cyto8-RETEV (SAESHKSGRETEV) was synthesized in solid phase using a Fmoc strategy (JPT).