Clone c29f4

Lung fibroblasts, which express a low level of swine leukocyte antigen (SLA) class I [49 ], were obtained as a kind gift from Dr. Jin-Hoi Kim, Konkuk University. The cells were seeded on glass coverslips (Warner Instruments, CT, USA) in a 6-well plate at a density of 1.2 × 10⁵ cells/well. On day 2, approximately 80% confluent cells were transfected with expression constructs, pCMV-HA, pCMV-B2M, or pEGFP, as described above. After 24 h, cover slips with attached cells were washed with cold phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were washed with cold PBS before blocking with 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cells were incubated with rabbit monoclonal antibodies specific to HA tags (dilution 1:250, clone C29F4; Cell Signaling Technology, MA, USA) or mouse monoclonal antibodies specific to SLA class I heavy chains (dilution 1:200, clone JM1E3; AbD Serotec, OX, UK) for 2 h at room temperature. After rinsing with cold PBS, cells were incubated with the goat anti-mouse/anti-rabbit antibody (1:500) conjugated with Alexa 568 (Invitrogen) for 1 h at room temperature, and the coverslips were mounted with a drop of Vectashield (with DAPI) (Vector, CA, USA) on a microscope slide. The cells were visualized using a fluorescence microscope DP72 (Olympus, Tokyo, Japan).

Details on processing human and mouse retina samples are described in supplementary material, Supplementary materials and methods. A rabbit monoclonal anti‐HA antibody (catalog # 3724S, clone C29F4, 1:3000; Cell Signaling Technology, Danvers, MA, USA) was used to detect the N‐HA‐tagged CLRN1 protein; an anti‐CLRN1 rabbit polyclonal antibody (catalog # 26630‐1‐AP, 1:2000; Proteintech, Rosemont, IL, USA) was used to detect human CLRN1; and mouse monoclonal anti‐alpha tubulin (catalog # T5168, clone B‐5‐1‐2, 1:5000, Millipore Sigma, Burlington, MA, USA) served as a loading control antibody.