**2. Materials and Methods**

### *2.1. Measuring Electrical Function in the Ter349Glu Rhodopsin Knock-in Mouse by Electroretinogram (ERG)*

Wild-type (+/+, WT), Ter349Glu rhodopsin heterozygous and homozygous mice at one month of age were dark-adapted overnight (O/N). The following day, the mice were anesthetized using ketamine/xylazine (14.3 mg/mL ketamine/2.8 mg/mL xylazine in PBS, pH7.4), and ERGs were performed. Dark-adapted flashes (505 nm stimulus) of varying intensities were achieved using neutral density (ND) filters of nominal optical densities (OD) 4.8, 1.4, 3.6, 2.4, 1.8, 1.2, 0.6, and 0.0. A dark-adapted green camera flash performed with no attenuation. Data were analyzed using Labview and IgorPro software.

### *2.2. Monitoring the Ter349Glu Rhodopsin Knock-in Mouse Retina for Vascular and Laminar Abnormalities*

WT and Ter349Glu homozygous mice at 4 weeks of age were anesthetized using ketamine/xylazine (14.3 mg/mL ketamine/2.8 mg/mL xylazine in PBS, pH7.4). The mice were subsequently examined by optical coherence tomography (OCT) (Bioptigen 840 nm Spectral Domain-OCT, Durham, NC, USA) to measure retinal thickness and assess for structural anomalies. After OCT, the mice were injected intraperitoneally with 100 μL of 4% fluorescein, and the retinas were imaged approximately 1 to 2 minutes post-fluorescein injection by fluorescein angiography (FA), using a 488 nm lamp co-equipped with a Micron III digital microscope (Phoenix Laboratories, Mukilteo, WA, USA) to assess the retinal vasculature for abnormalities including neovascularization, retinal hemorrhage, and vessel alterations.

### *2.3. Immunohistochemical Survey for Inflammatory Markers in the C-Terminal Mutant Rhodopsin Knock-in Mouse Retina*

Whole eyes from WT, Ter349Glu rhodopsin heterozygous and homozygous mice 4, 8, and/or 12 weeks of age were fixed in 4% paraformaldehyde (PFA) in PBS, pH 7.4, overnight at 4 ◦C, cryoprotected in 30% sucrose in PBS, pH 7.4, frozen in optimal cutting temperature medium, and cryosectioned into 10 μm-thick sections. After washing away the medium, sections were prepped for immunolabeling using heat-mediated antigen retrieval in 10 mM sodium citrate, pH 6.0, with 0.05% Tween-20 for 1 h. After cooling, the slides were washed in PBS and subsequently blocked in 10% goa<sup>t</sup> serum/5% BSA/0.5% Triton X-100 in PBS for 1 h at RT. Following blocking, labeling for markers of retinal inflammation including activated macrophages (F4/80), phosphorylated STAT3 (pSTAT3), and suppressor of cytokine signaling 3 (SOCS3) was performed using fluorescent immunohistochemistry. For both pSTAT3 (Cell Signaling Technology, Danvers, MA, USA) and SOCS3 (abcam, Cambridge, United Kingdom) labeling, a goa<sup>t</sup> anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP) was allowed to bind the primary antibodies, and Cy3-Tyramide Signal Amplification (TSA, Perkin Elmer, Waltham, MA, USA) was used to visualize the proteins. Briefly, TSA uses the peroxidase activity of HRP to generate reactive oxygen species from peroxide. These reactive oxygen species then oxidize and covalently bind the Cy3-conjugated tyramide molecule to the nearby proteins including the antigen/antibody complex, thus amplifying the signal. Macrophage labeling was performed using an antibody to F4/80-antigen (Bio-Rad, Hercules, CA, USA) followed by a goa<sup>t</sup> anti-rat IgG secondary antibody conjugated to AlexaFluor488 (Invitrogen, Carlsbad, CA, USA). All sections were co-labeled for rhodopsin using B6-30N or K62-82 (provided by W. Clay Smith, University of Florida, Gainesville, FL, USA) and goa<sup>t</sup> anti-mouse IgG1 or IgG3 conjugated to AlexaFluor488 (Invitrogen). Labeling for Müller cells was performed using an antibody against glial fibrillary acidic protein (GFAP, EMD Millipore, Burlington, MA, USA) followed by goa<sup>t</sup> anti-mouse secondary IgG1 conjugated to AlexaFluor647 (Invitrogen). All images were captured as Z-stacks and expressed as maximum intensity projections.

### *2.4. Analysis of Retinal Citrullination in the Ter349Glu*/*Ter349Glu Rhodopsin Mouse Retina by Fluorescent Immunohistochemistry*

Whole eyes from 10- to 12-week-old mice were fixed in 4% PFA in PBS, pH 7.4, overnight at 4 ◦C, cryoprotected in 30% sucrose in PBS, pH 7.4, frozen in optimal cutting temperature medium, and cryosectioned into 10 μm-thick sections. After washing away the medium, the sections were treated using heat-mediated antigen retrieval by boiling in 10 mM sodium citrate, pH 6.0, with 0.05% Tween-20 for 1 h. After cooling, the slides were washed in PBS and subsequently blocked in 10% goa<sup>t</sup> serum/5% BSA/0.5% Triton X-100 in PBS for 1 h at RT. Following blocking, the sections were incubated with primary antibodies against peptidyl arginine deiminase 4 (PAD4, ProteinTech, Rosemont, IL, USA) and citrullinated peptides (Clone F95, EMD Millipore, Burlington, MA, USA) O/N at 4 ◦C. After washing in PBS, the slides were probed using goa<sup>t</sup> anti-rabbit IgG conjugated to AlexaFluor488 (Invitrogen) and goa<sup>t</sup> anti-mouse IgM conjugated to AlexaFluor555 (Invitrogen), and the nuclei were labeled with DAPI (Invitrogen). After washing in PBS, the slides were mounted using Prolong Diamond Anti-Fade Mountant (Invitrogen) and imaged using a Zeiss 710 Laser Scanning Confocal Microscope (Zeiss, Oberkochen, Germany). All images were captured as Z-stacks and expressed as maximum intensity projections.
