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ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells

Keywords: intersectin , Ruk/CIN85 , Cbl-b , clathrin-coated pits , immunofluorescence

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Abstract:

Aim. Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b and are targeted to clathrin-coated pits in MCF-7 cells

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