Fitc anti mouse igg2a

293T cells were transfected with lipofectamine LTX reagent (ThermoFisher) with various full-length constructs of PILRA variants (G78 (AD risk), R72A, F76A, G78R, S80G, Q140A and S141A). After 48 hours, the transfected cells were harvested and incubated with soluble mIgG2a-tagged ligand, NPDC1-mFc at 50 μg/ml (as described above) for 30 minutes on ice. Cells were then washed and stained with 1 μg/ml chimeric anti-PILRA antibody (mouse Fc region is substituted to human IgG1 backbone on anti-PILRA antibodies [31 (link)]) on ice for 30 min followed by APC-conjugated mouse anti-human IgG (BD Pharmingen Cat.No. 550931) and FITC anti-mouse IgG2a (BD Pharmingen Cat. No. 553390) secondary antibodies according to manufacturer’s instruction. PILRA-transfected 293T cells were examined by flow cytometry for binding of NPDC1 by measuring the frequency of APC and FITC double-positive cells. Double positive cells were gated on the WT sample and than the gates were overlaid on subsequent samples to maintain the same cell population throughout the experiment. For each PILRA variant, the mean percentage of the number of cells binding to NPDC1-mFC relative to the wild type PILRA binding for each experiment was calculated.

In the inverse experiment, 293T cells were transfected with lipofectamine LTX reagent [ThermoFisher] with known full-length PILRA ligand (NPDC1, HSV-1gB and PIANP) and predicted ligand constructs (SORCS1, APLP1 and C4A) (described above). After 48 hours, the transfected cells were harvested and incubated with soluble mIgG2a-tagged variants of PILRA (G78 (AD risk), R72A, F76A, G78R, S80G) (described above) 50 μg/ml for 30 min on ice. Cells were then washed and stained with FITC anti-mouse IgG2a (BD Pharmingen Cat. No. 553390) secondary antibody according to manufacturer’s instruction. PILRA ligand-transfected 293T cells were examined by flow cytometry for binding to PILRA variants by measuring the frequency of FITC-positive cells. The percentage of mean fluorescence intensity (MFI) of PILRA-mFC binding on ligand-transfected cells relative to the wild type PILRA binding for each experiment was calculated.