Anti his hrp

For competition ELISAs, experimental conditions and solutions, not otherwise specified, were identical to the ones described above. Refer to each figure legend for the cells used and infection conditions (MOI and time postinfection) that generated the different supernatants tested. For CTLA4-CD80/CD86, CD28-CD80/CD86, and PD-L1–CD80 competition assays, 100 μl of CTLA4-Fc, CD28-Fc, and PD-L1–Fc was coated at 0.25 μg/ml (CTLA4), 2 μg/ml (CD28), or 1 μg/ml (PD-L1). Samples were added and diluted (2-fold serial dilution) in blocking solution containing a constant concentration of either CD80-Fc (biotinylated or not; from 10 to 250 ng/ml) or CD86-Fc (biotinylated or not; from 100 to 500 ng/ml), depending of the interaction. Either anti-His-HRP (Qiagen) at 1/5,000 or streptavidin-HRP (Southern Biotech) at 1/1,000 was used as a conjugated reagent. The plates were further treated, and results were analyzed as described above.

AFP1-His and AFP2-His expressed and purified from N. benthamiana were coupled to NTA-agarose beads in binding buffer (25 mM Tris-HCl, 0.3 M NaCl, and 0.1% NP-40, pH 7.5). The beads were then incubated with the supernatant collected from SG200Δrsp3-derived strains constitutively expressing secreted Rsp3-HA variants for 4 h at 4 °C. The beads were washed with binding buffer and proteins bound were removed by boiling in sample buffer and subjected to western blotting analysis using anti-His-HRP (1:8000 dilution, Qiagen Cat#34460) and mouse monoclonal anti-HA antibody (1:10,000 dilution; Sigma Cat#H9658). Horseradish peroxidase-conjugated anti-mouse or IgG (1:10,000 dilution; Sigma Cat#H9658) was then used as secondary antibody.