Figure 2. GDNF, NRTN, and ARTN bind... | Rockefeller University Press

Figure 2.

$Figure 2. GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The Kds were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. Kon is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with heparinase III (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.$

GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K_ds were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K_on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of ¹²⁵I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with heparinase III (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with ¹²⁵I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.