Substance P Increases Cell-Surface Expression of CD74 (Receptor for Macrophage Migration Inhibitory Factor): In Vivo Biotinylation of Urothelial Cell-Surface Proteins
Figure 3
Expression of
CD74 and MIF on urothelial bladder surface. (a) Total biotinylated protein. Isolated
urothelial cells were lysed, biotinylated proteins were isolated by avidin
agarose affinity chromatography, separated by 4–12% bis tris
acrylamide gels electrophoresis,
transferred to PVDF and biotin containing protein bands visualized with
strepavidin-HRP only, no antibodies were used.
P: indicates precipitation of proteins with avidin agarose, D: indicates
detection with SA-HRP only, no antibodies were used. Lanes 1–6, saline-treated
animals; lanes 7–12 SP-treated
animals. (b) Immunoprecipitation of biotinylated CD74 or MIF. Isolated
urothelial cells were lysed, 1 mg of total biotinylated proteins was isolated by avidin
agarose affinity chromatography and biotinylated CD74 or MIF protein was
immunoprecipitated using appropriate antibodies. Precipitates were separated by
denaturing-reducing SDS PAGE and CD74 or MIF protein detected by
strepavidin-HRP. P: indicates precipitation of proteins with MIF antibody (left
panel) or CD74 antibody (right panel). D: indicates detection with SA-HRP only
(left and right panel), no antibodies were used. Lanes 1, 2 saline-treated
animals (number 4 and 5 from Figure 3(a)), lanes 3, 4 SP-treated animals
(numbers 9 and 10 from Figure 3(a)). G: indicates immunoprecipitation with
GAPDH antibody documents that only biotinylated proteins were
immunoprecipitated, N: indicates immunoprecipitation with nonspecific goat IgG
documents antibody specificity. Lines and numbers to the far left indicate the
position of molecular weight markers. Asterisk indicates the location of 76 kDa band. Arrow indicates the location of 12 kDa uncomplexed MIF. (c) Coimmunoprecipitation
of cell-surface MIF/CD74 complexes. Isolated urothelial cells were lysed, 1 mg
total protein was used to purify biotinylated proteins by avidin agarose
affinity chromatography. Biotinylated MIF proteins were precipitated with an
anti-MIF antibody. Precipitates were separated by denaturing-reducing SDS PAGE
and CD74 protein containing bands identified using anti-CD74 antibody followed
by an antigoat-HRP. P: indicates precipitation of proteins by MIF antibody. D:
indicates detection with CD74 primary antibody and antigoat HRP secondary
antibody. Upper panel saline-treated animals, Lanes 1, 2, 3, 4, (numbers 1, 2,
3, and 6 from Figure 3(a)). Lower panel SP-treated animals, Lanes 1, 2, 3, 4,
(numbers 7, 8, 11, and 12 from Figure 3(a)). N: indicates immunoprecipitation
with nonspecific goat IgG documents antibody specificity.
