Fig. 10. Targeting of AMF to the ER and CTX to the Golgi apparatus is tyrosine kinase dependent. NIH-3T3 cells were pretreated with genistein (100 µg/ml) for 30 minutes during ligand incubation (A-I) or left untreated (J-L). Cells were pulse labeled with FITC-CTX for 30 minutes at 37°C (A,D) and labeled with monoclonal anti-GM130 (B) or anti-TfR antibodies (F) following by Alexa 568 anti-mouse secondary antibody. The merged confocal images present FITC-CTX in green and either GM130 (C) or TfR (F) in red and colocalization in yellow (C,F). Genistein selectively inhibits CTX delivery to the Golgi but not to TfR-positive endosomes. Alternatively, NIH-3T3 cells were pulse labeled with AMF-FITC for 5 minutes at 37°C. AMF-FITC was revealed with rabbit anti-FITC followed by Alexa 488 anti-rabbit antibodies (G,J) and the smooth ER labeled with anti-AMF-R followed by rhodamine-red-X anti-rat IgM antibodies (H, K). The merged confocal images present AMF-FITC in green and AMF-R in red and colocalization in yellow (I,L). The image of AMF endocytosis in the presence of genistein (G) was acquired at the same intensity level as the control in the absence of genistein (J), clearly demonstrating that genistein inhibits AMF delivery to the smooth ER. Bar, 8 µm.

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