Primary Antibodies Used

To probe for PAD4 and retinal citrullination, rabbit anti-PAD4 (12373-1-AP; Proteintech Group, Inc., Rosemont, IL, USA) and mouse anti-citrullinated peptide clone F95 (MABN328; MilliporeSigma, Burlington, MA, USA) were used, respectively. Ascertaining PAD2 expression proved more challenging. We attempted to obtain the monoclonal antibody cited by Bonilha et al.,¹⁰ but this was unavailable for our use. We then tried other commercially available antibodies, but none tested demonstrated sufficient, if any, binding of PAD2. To circumvent these challenges, a rabbit anti-PAD2/4 cross-reactive antibody was used to label for both PAD2 and PAD4 (12110-1-AP; Proteintech) in both WT and PAD4KO animals. Cross-reactivity was tested by western blot on purified recombinant PAD2- glutathione-S-transferase (rPAD2-GST) and purified rPAD4-GST fusion proteins (abcam, ab125591, and ab126910, respectively). Considering the absence of PAD4 in the KO mice (see “Results”), any residual PAD labeling in PAD4KO animals using this antibody was interpreted as attributable to PAD2, while labeling in WT animals was attributed to both PAD2 and PAD4 (total PAD labeling). Housekeeping protein GAPDH served as the loading control, and mouse anti-GAPDH (abcam, ab8245) antibodies were used for this purpose. Other antibodies used for IHC include rabbit anti-β-tubulin (abcam, a179513), mouse anti-glutamine synthetase (GS) (MAB302; MilliporeSigma), and chicken anti-neurofilament H (NFH) (PA1-10002; Thermo Fisher Scientific, Inc., Waltham, MA, USA).