Journal: Molecular Biology of the Cell
Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization
Figure Lengend Snippet: BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p
Article Snippet: After washing twice with 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and boiling in 2× SDS-PAGE sample buffer for 10 min, samples were subjected to 8% SDS-PAGE and immunoblotting using anti-myc antibodies (Covance Research Products).
Techniques: Binding Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Stable Transfection, Labeling, Plasmid Preparation