Figure 6.

Immunofluorescence copatching reveals a ligand-mediated increase in BMP receptor heterocomplex formation at the cell surface. COS7 cells were cotransfected with myc–BR-Ia and HA–BR-II (A and B), HA–BR-Ib and myc–BR-II (C and D), or myc–BR-Ia and HA–BR-Ib (E and F). Live cells were labeled in the cold consecutively by a series of antibodies to mediate patching and fluorescent labeling, as described in MATERIALS AND METHODS. After this labeling procedure, HA-tagged receptors are labeled by Cy3 (red), myc-tagged receptors are labeled by FITC (green), and patches containing both tags appear yellow when the two fluorescent images are superimposed. The labeling specificity is demonstrated by the existence of separately labeled patches (only red or only green) in the coexpressing cells and by the exclusively green or exclusively red staining of cells singly transfected with either myc–BR-II or HA–BR-II alone and labeled by both antibody sets (see Figure 7E, insets). (A, C, and E) No ligand added. (B, D, and F) Incubation with 10 nM BMP-2. The ligand was added together with the normal goat IgG before copatching (for 1.5 h) and kept in during all successive incubations. Bar, 10 μm.