PMID: 7537292May 1, 1995Paper

Ultrastructural localization of ras-related proteins using epitope-tagged plasmids

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
D RobertsonP Monaghan

Abstract

To determine the ultrastructural distribution of H-ras, the rho proteins rho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-binding protein family), we used cDNA expression plasmids in which a short sequence coding for the epitope recognized by the anti c-myc monoclonal antibody 9E10 has been inserted at the N-terminus. Each of the expressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence labeling with 9E10 imaged by confocal microscopy. For ultrastructural localization of these tagged proteins, a method was devised to process microinjected cells in situ into low-temperature resin. The proteins were localized on the sections using 9E10 detected with colloidal gold conjugates. Ha-ras protein was localized almost exclusively on the cell membranes. Rho-A and rho-C were predominantly associated with the submembraneous actin network, and rho-B was found in association with multivesicular bodies. Rac1 protein induces the formation of large pinocytotic vesicles and was detected on the cytoplasmic face of...Continue Reading

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