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. 2022 Jan 19;298(3):101605. doi: 10.1016/j.jbc.2022.101605

Figure 4.

Figure 4

Epitope competition study.A, the Ni-NTA affinity-purified His-tagged CysR-CTLD1, CysR-CTLD3, and CysR-CTLD8 domain fragments were heated for 5 min at 100 degrees in SDS-sample buffer (no β-ME) and resolved by 4 to 20% SDS-PAGE. The gel was stained with Coomassie blue overnight. B, the PLA2R-Ab positive sera (20 μl each) were individually mixed with 30 μg BSA, 30 μg CysR-CTLD8, or 30 μg CysR-CTLD1 fragments in 100 μl TBS (pH 7.4) and incubated on a rotating shaker for 2 h at room temperature. The mixture was then applied on the membrane blocked in 2 ml TBSTM buffer and incubated for 2 h at room temperature. The level of each protein sample on the blot was verified by the Anti-PLA2R-Ab. Each experiment was performed at least three times. -, no blocking reagents; Anti-PLA2R-Ab, rabbit anti-human PLA2R polyclonal antibody; BSA, bovine serum albumin; CTLD, C-type lectin-like domain; CysR, cysteine-rich domain; CysR-CTLD1-T, CysR-CTLD1 triple domain truncation; CysR-T, CysR domain truncation; PLA2R, phospholipase A2 receptor; PLA2R-F, PLA2R full-length.