Fig 2: Novel multiplex tyramide signal amplification immunofluorescence protocol. Multiplex tyramide signal amplification can be organized in a flexible protocol divided into three rounds of staining separated by heat‐induced antigen retrieval and imaging. Optimized block includes a combination of TrueBlack, Image‐iT FX Signal Enhancer, and 5% BSA. Anti‐human primary antibodies can be substituted for mouse‐specific or other species‐specific antibodies without requiring alterations to subsequent secondary antibody or tyramide reagents. Curing is an overnight process and imaging is followed by overnight coverslip removal before the subsequent antigen retrieval and blocking steps can begin. Round 2 contains an antigen retrieval step to allow for the usage of two primary antibodies both made in rabbit. Any of the three rounds may combine primary antibodies of the same species given that they are separated by an antigen retrieval step and are paired to different tyramide fluorophores. It is important to note that once a specific tyramide fluorophore has been used that channel may no longer be used in following rounds. BSA, bovine serum albumin; DAPI, 4′,6‐diamidino‐2‐phenylindole; HRP, horseradish peroxidase; PSA, prostate‐specific antigen; PSMA, prostate‐specific membrane antigen

Fig 3: MS/MS Fragmentation spectrum for ASN-336 PSMA glycopeptides after PNGase-F cleavage of the glycans in the presence of light and heavy water. Spectrum showing b and y ions from the PSMA glycopeptide with sequence VPYNVGPGFTGN*FSTQK in the presence of 18O heavy water; the deamidation results in an increase in mass of the aspartic acid by 2.98 Da in the upper panel. A similar spectrum showing b and y ions after ASN-336 deamidation in the presence of normal 16O water with a mass increase of 0.98 Da of the aspartic acid is shown in the lower panel.

Fig 4: Representative Western blot analysis showing the expression levels of CD63, the common exosome marker, PSMA and caveolin-1 in PCa and BPH samples of plasma-derived Exos and Ponceau S staining as a loading control (a). The relative protein levels of PSMA and caveolin-1 in the plasma Exos of PCa and BPH patients were determined by Western blot analysis and normalized to Ponceau S (relative protein level, y-axis) (b). A Mann–Whitney U test or a Kruskal–Wallis test was used to determine statistical significance. Data are shown as interquartile ranges. The horizontal line in the boxes represents the median values ± SD. Pca—prostate cancer; BPH—benign prostatic hyperplasia; PSMA—prostate-specific membrane antigen, *—statistical significance.

Fig 5: Fractionation of the CM derived from LNCaP cells. (A) Confocal images of HUVECs cultured with each fraction of CM derived from LNCaP cells. Each fraction was diluted to half of its concentration with EBM‐2 or suspended with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. The PSMA‐positive HUVECs were shown by arrowheads. Bars: 100 µm. The representative images from three independent experiments were shown. (B) Confocal images of HUVECs cultured with 10,000g pellet fractions of CM derived from LNCaP cells. The 10,000g pellet fraction was labeled with a membrane marker dye, before the suspension in EBM‐2 medium. The labeled 10,000g pellet was cultured with HUVECs for 6 h, and cells were subjected to immunofluorescence staining for PSMA. Bars: 10 µm (left) and 2 µm (right; magnified images of squares in left images). The representative images from three independent experiments were shown. (C) Western blots of 10,000g pellet fraction of CM derived from LNCaP cells. The lysates from HUVECs and LNCaP cells were used as negative and positive controls of PSMA expression, respectively. The representative blot data from three independent experiments were shown. (D) Confocal images of HUVECs cultured with the CM derived from PC3 cells that stably express Myc‐PSMA or PSMA‐myc. The CM was diluted in half with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. The representative images from three independent experiments were shown. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com]