Fig. 2. In vitro citrullination and glycation assays.

a NCPs were first citrullinated by PAD4 followed by a 5 mM MGO treatment at 37 °C overnight. The reactions were separated on a native gel followed by a western blot analysis. (b) NCPs were pretreated with 5 mM MGO for short (S, 6 h) or long (L, overnight) periods and then incubated with PAD4, or concurrently (C) incubated with both the enzyme and MGO for the same time. c PAD4 was incubated with glycated nucleosomal arrays after short (S) or long (L) treatment with MGO, or co-incubation (C). Error bars represent the standard deviation from three different experiments. Data are presented as mean values ± SEM. d Dot blot analysis of MGO glycation and PAD4-mediated deglycation on H3 N-terminal peptide substrate. The H3 peptide sequence is NH₂-ARTKQTARKSTGGKAPRK(Bio)A-COONH₂, and the biotin signal (imaged by Atto 680-Streptavidin) was used as loading control. e LC-MS analysis of modified and unmodified H4 peptides (λ = 214 nm): (i) H4-R3(1-6) peptide, (ii) H4-Cit3(1-6) peptide, (iii) MGO-treated H4-R3(1-6), (iv) MGO-glycated H4-R3(1-6) followed by PAD4 treatment in H2¹⁸O. The H4 synthesized peptide sequences are AcNH-SGRGK(Bio)G-COONH₂ and AcNH-SGCitGK(Bio)G-COONH₂.