Fig. 6. GFP-tagged Y237A-mNTR2 and phosphotyrosine staining in transfected COS-7 cells. Transiently transfected COS-7 cells were either untreated (A-C) or treated with 100 nM NT at 37°C for 5 minutes (D-F) or 15 minutes (G-I) prior to fixation. GFP receptor fluorescence (A,D,G) and antiphosphotyrosine labeling revealed with Cy-5 conjugated donkey anti-mouse antibody (B,E,H) are visualized as described in Materials and Methods. Overlays of GFP-Y237A-mNTR2 and phosphotyrosine fluorescent labeling are shown in C, F and I. The yellow color indicates colabeling. F' and I' are enlargements of boxes in F and I images. Arrowheads show clusters of mutated receptors. Bar, 10 µm.