Figure 6.

Proteolytic cleavage of galectin-3 in prostasomes.

A: Human testis, epididymis, vas deferens, seminal vesicle, prostate, sperm, prostasome extracts, clarified seminal plasma, and PSA-cleaved recombinant galectin-3 were evaluated for galectin-3 immunoreactivity by immunoblot analysis. An ~16 kDa immunoreactive band indicative of cleaved galectin-3 was detected in the prostate and prostasomes extracts, seminal plasma, and the PSA-treated galectin-3 sample. B: Biotinylated galectin-3 was cleaved with purified PSA or prostasomes. Galectin-3 cleavage products were visualized by electroblot analysis with streptavidin-HRP. C: The galectin-3 cleavage assay was performed with prostasomes that were pre-incubated with 5 mM PMSF, 50 mM ZnCl₂, 1 mM TLCK, or 20 mM 1,10-phenanthroline. D: The galectin-3 cleavage assay was performed with untreated prostasomes and prostasomes that were pre-incubated with PSA neutralization antibody or irrelevant antibody. Untreated galectin-3 was included as a negative control for electroblot analysis. E: Percent cleavage of galectin-3 was calculated for the neutralized PSA reaction and controls in D. Each bar represents the mean of three independent experiments; * p-value <0.05, ** p-value <0.005.