**3. Differential subcellular localization of AGR2**

The contrasting localization of eAGR2 and iAGR2 is well illustrated by the bladder. The entire urothelium is positive for AGR2 expression as shown by immunostaining [18] (**Figure 1**). If AGR2 is secreted, then a substantial amount of this protein would be found in voided urine. In fact, little AGR2 is released by normal iAGR2+ urothelial cells as detected by ELISA [18]. This result was supported by urine proteome database queries that AGR2 was not listed in the *UrinePA-PeptideAtlas* of 2,500 proteins profiled by large-scale proteomics [29], and in the core urinary proteome of 587 proteins obtained from healthy people [30]. In contrast, we found that AGR2+ bladder cancer cells secreted AGR2. Urine from a bladder cancer patient was scored 7.5-fold higher than buffer control for AGR2. Five of 20 bladder cancer patient urine in one cohort were tested positive (AUC = 0.75), which matched the 25% bladder cancer being positive for AGR2 expression [18]. Our urine assay also detected AGR2 secreted by prostate cancer producing a similar AUC [31]. For serum, level of AGR2 is near background in healthy people [32]. Query of the *PeptideAtlas* database yielded very low AGR2 signature peptide counts. However, when sera of five prostate cancer patients were analyzed for AGR2, there was a strong correlation, *R*<sup>2</sup> = 0.93, found between levels of AGR2 (in pg/ml) and those of PSA (in ng/ml) [32]. This result would not be possible if there was a basal level of blood AGR2 arising from possible secretion by AGR2+ cells of normal tissue such as the urothelium and lung epithelium into the circulatory system [32].
