GRP78-MIF complexes in the bladder.

<p>A) Representative results of GRP78 “pull-down” experiments where GRP78 complexes were isolated from bladder homogenates by immunoprecipitation and then MIF (left side of figure) or A1-I3 (right side of figure) western blotting was carried out to identify complexes. Recombinant MIF was loaded (1^st lane) as positive control. Note that GRP78-MIF complexes (marked by ## at 12 kDa, lane marked Sal = Saline) increased after substance P treatment (lane labeled SP). A1-I3 western blotting (right side) showed several fragments associated with GRP78, as expected, since GRP78 is a receptor for A1-I3. Also note that while 12 kDa MIF was not detected, several high-molecular weight fragments contained both MIF and A1-I3 (130 and 80 kDa), as reported previously<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005835#pone.0005835-Vera3" target="_blank">[9]</a>. B) GRP78-MIF complexes at the urothelial cell-surface. Urothelial cell-surface proteins were biotinylated, isolated and then GRP78 biotinylated complexes were “pulled down” using GRP78 antibodies. The resultant protein complexes were then electrophoresed and biotin activity was detected using biotin-horseradish peroxidase-chemiluminescence. A faint band of biotinylated (cell-surface) proteins at approximately 78 kDa (corresponding to GRP78 molecular weight) were “pulled down” by GRP78 immunoprecipitation in saline animals, while this band increased in intensity in substance P-treated animals (asterisk). Therefore, these results show that GRP78-MIF urothelial cell-surface complexes increase after substance P-treatment.