Recombinant gfp

Supernatants from cultured CD4+ T cells were harvested after 48–72 hours of culture, as described above. Analytes were probed for using the following kits: IL-4 (R&D, Invitrogen), IL-5 (Invitrogen), IL-10 (Invitrogen), IL-13 (Invitrogen), IFN-γ (Invitrogen), and amphiregulin (R&D). For serum immunoglobulin ELISAs, serum was isolated from naive mice or infected mice at the indicated timepoints and diluted 1:1000 in PBS. Plates were coated with 1 ug/mL of the appropriate anti-Ig antibody (Bethyl Laboratories, Inc.), and immunoglobulins were detected using 1 ug/mL of horseradish peroxidase (HRP)-conjugated anti-Ig antibody (Bethyl Laboratories, Inc.). For anti-GFP ELISAs, serum was isolated from naive mice or infected mice 14 days post-infection and diluted as indicated in PBS. Plates were coated with 10 ug/mL recombinant GFP (Abcam), and GFP-specific IgM and IgG were detected using 1 ug/mL of HRP-conjugated anti-IgM/IgG antibody (Jackson Immunoresearch).

Proteins were transfected into yeast cells using the Xfect™ Protein Transfection Reagent (Takara Bio Inc., Shiga, Japan), according to the Manufacturer’s instruction with modifications. Briefly, yeast cells (4.0 × 10⁷ cells/ml) suspended in PBS (pH 7.4) or 10 mM MES (pH 6.0) were mixed with the Xfect reaction buffer along with proteins, preincubated at room temperature for 30 min, and then washed with PBS to remove the reaction reagent. Proteins used were 4 µg β-Gal (accessory in Xfect reagent), 46 µg recombinant GFP (Abcam, Cambridge, CB2 0AX, UK), 5 µg Alexa Fluor 488 goat anti-rabbit IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), and 50–70 µg recombinant NLS-TaqI (prepared in this study; see below). In experiments shown in Fig. 1b, protein/Xfect complexes absorbed on the extracellular membrane or cell wall were digested by 1% trypsin/PBS treatment at 37 °C for 5 min^{32 (link),40 (link)}.