Construction and identification of RFP-GFP-Ckap4 knock-in transgenic mice. (A) Schematic illustrating the construction of the ER-phagy reporter Ckap4-KI using a knock-in mouse model. The tandem fluorescent protein RFP-GFP was inserted at the 5′ end of the first exon of the Ckap4 gene on chromosome 10. (B) Genotyping of transgenic Ckap4-KI mice. Mouse genotypes were identified using PCR with three pairs of primers, as detailed in the materials section. “WT” denotes wild type, “Het” indicates heterozygote. (C) Western blot analysis demonstrating the expression of the Ckap4-KI reporter in knock-in mice. The expression of the Ckap4-KI reporter was assessed in various tissues of both wild-type and knock-in mice. Double red asterisks (**) indicate the fused RFP-GFP-CKAP4 protein, detected by anti-CKAP4 and anti-RFP antibodies. Single red asterisks (*) denote endogenous CKAP4 detected by anti-CKAP4 antibody. GAPDH and Tubulin served as internal controls. Tissues were harvested from 8-wk-old mice. “−” denotes WT, “+” represents heterozygotes. (D) Representative microscopy images illustrating the localization and expression level of the Ckap4-KI reporter in mouse testis tissue. Scale bar: 20 μm. See also Fig. S1. Source data are available for this figure: SourceData F1.