Fig 2: Elevated protein levels of AGR2 in canine MMT tissues compared with paired normal mammary tissues. Tissue homogenates (20 µg proteins each sample) of MMT (denoted M), paired with benign mammary tumor (denoted B) and normal mammary gland tissues (denoted N) collected from the same patient, were analyzed by immunoblotting using an antibody specific to AGR2. GAPDH was used as an internal control. Representative results of nine cases (No. 1 to No. 9) were shown in (A). AGR2 protein levels in individual samples were determined by calculating the AGR2-to-GAPDH ratio. (B) Comparison of AGR2 protein levels between tissue types. Statistical significance was determined by Mann–Whitney U test. (C) Pairwise comparison of AGR2 protein levels between MMT and paired normal mammary gland tissues of the same patient. Lines represent individual patients. Statistical significance was determined by two-tailed paired t-test. ** p < 0.01; *** p < 0.001.

Fig 3: Establishment of a competitive ELISA for detection of serum eAGR2 in MMT dogs. (A) Scheme for the established competitive ELISA. (B) Identity of recombinant canine AGR2 (rcAGR2) was confirmed by immunoblotting using antibodies specific to AGR2 and 6×His tag, respectively. (C) A standard curve with fourfold serially diluted rcAGR2 proteins (8000, 2000, 500, 125, and 31.25 ng/mL) was set for the competitive ELISA. Data are presented as the mean values ± SD across 10 assays. (D) Precision of the competitive ELISA. The intra-assay coefficient variant (CV), inter-assay CV, and the detection sensitivity were determined across 10 assays. (E) Evaluation of serum influence on eAGR2 detection in the ELISA. A spike recovery test was performed by detecting rcAGR2 spiked at indicated concentrations into canine serum containing undetectable eAGR2. The recovery rate (%) of individual spike rcAGR2 was calculated as indicated.

Fig 4: Detection of AGR2 expression in canine MMT cells and conditioned media. (A) Intracellular localization of AGR2 in canine MMT cell lines, DMGT, and CF41.Mg. AGR2 expression was verified by immunofluorescence staining using an AGR2-specific antibody. DAPI staining indicated the nuclei. Magnification, ×40. (B) Detection of AGR2 in cell lysates of canine MMT cell lines. Cell lysates (30 µg proteins per sample) were analyzed by immunoblotting with an AGR2-specific antibody. (C) Detection of extracellular AGR2 (eAGR2) in serum-free conditioned media of canine MMT cells. Cells were grown in serum-free media for one day, and the conditioned media were harvested and subsequently concentrated. The resulting conditioned media (50 µg proteins per sample) were analyzed using a competitive ELISA established for eAGR2 detection.