RP - Immunofluorescence assays for EGFR and TfR (»Add to Infobox)

Di Fiore Pier Paolo, Polo Simona

Pier Paolo Di Fiore's Group, Simona Polo's group

This assays allows a qualitative analysis of EGFR and TfR internalization, unveiling defects in internalization and/or subsequent trafficking steps along the endocytic pathway. Colocalization of the fluorescently-labeled ligands with markers of the different endocytic compartments allows to follow the trafficking of both receptors along the endocytic route. TfR, once internalized in the endosomal/recycling compartment (rab11-positive), is recycled back to the plasma membrane. EGFR, once it reaches the endosomes, is either recycled back to the surface or targeted for degradation into the lysosome. In HeLa cells, after 15 min it localizes with endosomal markers (e.g. EAA1); at 30-60 min it reaches the lysosomes (Lamp-1/2) and the signal starts to disappear because of degradation. Treatment with lysosomal inhibitors (such as chloroquine) causes the accumulation of intracellular EGF/EGFR complex.

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Di Fiore Pier Paolo (Principal Investigator / Pier Paolo Di Fiore's Group)

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