Construction and identification of RFP-GFP-Reep5 knock-in transgenic mice. (A) Schematic for constructing the ER-phagy reporter Reep5-KI using a knock-in mouse model. Tandem fluorescent protein RFP-GFP was inserted at the 5′ end of the first exon of the Reep5 gene on chromosome 18. (B) Genotyping of transgenic Reep5-KI mice. Mouse genotypes were determined using PCR with three pairs of primers, as detailed in the materials section. (C and D) Western blot analysis demonstrating the expression of the Reep5-KI reporter in knock-in mice. The expression of the Reep5-KI reporter was assessed in various tissues of both wild-type and knock-in mice. Double red asterisks (**) indicate the fused RFP-GFP-REEP5 protein, single red asterisks (*) denote endogenous REEP5 identified by anti-REEP5 antibody, and single black asterisks (*) highlight the RFP fragments. GAPDH and Tubulin served as internal controls. Tissues were obtained from 8-wk-old mice. “−” denotes WT, “+” represents heterozygotes, and “++” indicates homozygotes. (E) Representative microscopy images illustrating the localization and expression level of the Reep5-KI reporter in mouse pancreas tissue. Homozygotes are denoted as “Hom.” Scale bar: 20 μm. See also Fig. S1. Source data are available for this figure: SourceData F2.