Figure. 5. Cross-reactive antibodies modify accessibility of PAD4 to protease cleavage at D³⁸⁸.

(A) IVTT wild type or D³⁸⁸→A mutated PAD4 (filled arrows) was incubated with 0, 5, or 50 nM GrB for 1 hour at 37°C and dominant fragments were detected (open arrows). (B) IVTT PAD4 or the negative control IVTT B23 was pre-incubated with no IgG, PAD4-only IgG (P4), or cross-reactive IgG (XR) prior to incubating with 50 nM GrB. (C) Proteins were visualized by radiography and “% Protection from GrB cleavage” was determined by densitometry. Representative data from 3 independent experiments performed with either 5 nM or 50 nM protease is compiled and shown. A two-tailed t-test was performed and a p-value<0.05 was considered significant.