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. 2021 Apr 9;296:100648. doi: 10.1016/j.jbc.2021.100648

Figure 4.

Figure 4

Identification of acrolein-specific innate epitopes. A, reverse-phase HPLC analysis of acrolein-treated NAK. The reaction was performed by incubating 10 mM NAK with 1 mM acrolein in PBS (pH 7.4) for 24 h at 37 °C. The reaction was monitored by the absorption maximum at 200 to 600 nm (solid line) and at 270 nm (dotted line). B, chemical structure of Nε-3-formyl-3,4-dehydropiperidino-Nα-acetyl-L-lysine (FDP-NAK) (peak a) and Nε-(3-methylpyridinium)-Nα-acetyl-L-lysine (MP-NAK) (peak b). C, dose-dependent production of IgM by FDP-NAK and MP-NAK in the PerC cells. D, dose-dependent differentiation of B-1a cells into the plasma cells by FDP-NAK and MP-NAK. E, antigen-binding activity of the IgM Abs produced by the cells that had been treated with the acrolein-specific adducts (FDP-lysine and MP-lysine). The PerC cells were treated with 0.5 mM NAK or 0.1 to 0.5 mM acrolein-lysine adducts (FDP-NAK and MP-NAK) for 72 h at 37 °C, and the culture supernatants were tested for the cross-reactivity with the control BSA or acrBSA. Differences were analyzed by Dunnett's test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; versus N/C (C and D); versus N/C within each IgM response to the same antigen (E). Abs, antibodies; acrBSA, acrolein-modified BSA; BSA, bovine serum albumin; IgM, immunoglobulin M; NAK, Nα-acetyl-L-lysine.