Pierce ip lysis

About 10 × 10⁶ COS-1 cells were transfected with 1 µg/1 × 10⁶ cells of the specific vector using Metafectene transfection reagent (Biontex). At 6 hpt, cells were infected with VV-T7 (MOI = 0.5 PFU/mL). At 16 hpi, cells were collected and lysed with 1 mL of Pierce IP Lysis (ThermoFisher Scientific) and protease and phosphatase inhibitors for 2 h on ice. Lysates were centrifuged and precleared with Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific). CD2v variants were immunoprecipitated using either Pierce Anti-c-Myc Magnetic Beads or anti-CD2v bonded to magnetic beads Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific), as indicated, at 4 °C with rotation overnight. Different immunoprecipitates were eluted and non-treated or treated with either endoglycosidases PNGase-F or Endo-H (New England BioLabs). Briefly, IP elution was denaturalized using denaturalized buffer at 100 °C for 10 min and then chilled on ice, centrifuged at 11,000 rpm, and incubated with PNGase-F or Endo-H (1 h at 37 °C). Finally, samples were analyzed by Western blot.

Blood samples were collected and centrifuged at 10,000× g for 2 min to separate serum. Serum samples were kept at −80 °C. Small pieces of ileum were snap-frozen and kept at −80 °C until protein extraction. To examine the cytokine levels, small parts of the ileum tissues (20–25 g) were collected in microtubes containing lysis buffer (Pierce IP Lysis, Thermo Fisher Scientific) and protease/phosphatase inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific) and homogenized using an electrical homogenizer (Bead mill 24, Fisher Scientific, USA). IgA and IgG levels were determined in serum using mouse IgA and IgG uncoated ELISA kits (Invitrogen, Vienna, Austria). The levels of IL-6, IL-10, IL-17A, and IL-23 were measured in ileum tissues using mouse-uncoated ELISA kits (Invitrogen, Vienna, Austria). All ELISA tests were performed according to the manufacturer’s instructions. Absorbance was read at 450 nm with a wavelength correction of 570 nm using a microplate reader (Bio-TEK Instruments, Winooski, VT, USA).