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Fig. 5. MT1-MMP is required for efficient ligand-induced internalization of PDGFRβ in VSMCs. (A) VSMCs on polymerized collagen I were fixed and permeabilized for immunofluorescence staining using antibodies against PDGFRβ and caveolin 1. PDGFRβ colocalized with caveolin 1 (caveolae) in both wild-type (+/+) and MT1-MMP-null (–/–) cells, but specific intracellular caveolin-1 structures did not contain PDGFRβ in MT1-MMP–/– VSMCs. (B) To detect internalization, quiescent VSMCs were labeled with anti-PDGFRβ antibodies for 1 hour at 4°C. Unbound antibodies were removed and the cells shifted to 37°C for 30 minutes in the presence of PDGF-BB (10 ng/ml). After blocking the anti-PDGFRβ antibodies that remained on the cell surface with unlabelled secondary antibodies, the cells were fixed, permeabilized and stained with anti-caveolin-1 antibodies followed by secondary Alexa-Fluor-488- and Alexa-Fluor-594-conjugated antibodies. The merged high-magnification image depicts colocalization between internalized PDGFRβ and caveolin 1 in yellow. Note the efficient PDGFRβ internalization in caveolin-positive structures of wild type (+/+) VSMCs in contrast to minor PDGFRβ internalization in MT1-MMP-null (–/–) cells.
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