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Figure 3:FACS analysis of cells with MAb to hAPRIL (Aprily-1).Method: HEK 293 cells were mock transfected or transfected with an expression plasmid enabling surface expression of a non-cleavable human APRIL. Cells (5×105) were incubated on ice for 30 min in 50 µl FACS buffer (PBS, 5% Fetal calf serum, 0.02% azide) containing 10µg/ml of Aprily-1 antibody. After washing in FACS buffer, FITC-conjugated antibody to mouse IgG was added. Cells were incubated on ice for 30 min., washed and analyzed by flow cytometry.
Figure 2: Detection of endogenous human APRIL with MAb to APRIL (Aprily-1) after IP with recombinant human BCMA:Fc.Method: Cell supernatants were first precleared with protein-A sepharose and then subjected to immunoprecipitation with rhBCMA:Fc (Prod. No. ALX-522-026) and protein-A sepharose. Beads were washed twice with PBS. Immunoprecipitated materials were eluted with 2.5mM glycine pH 2.5 for 10 min., neutralized with Tris buffer and run on an SDS-PAGE gel and analyzed with 2µg/ml Aprily-1 by Western blot. Note: In some migrations endogenous soluble APRIL resolved as a doublet around 20kDa (dendritic cells: DC). MegaAPRIL™, Soluble (human) (rec.) (Prod. No. ALX-522-035) served as a positive control.”””””
Figure 1: Western blot of total cell extracts from HEK 293 cells transfected with the indicated expression vector. Aprily-1 reacts specifically with human APRIL.Method: 10µg of protein was applied to the gel. Revealed with Aprily-1 (1µg/ml) and HRP-coupled anti-mouse secondary antibody (1:4’000).Note: The human APRIL construct used in this experiment is an uncleavable fusion protein between human BAFF (aa 1-132) and human APRIL (aa 93-233).
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RUO – Research Use Only |
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