Fig. 7. Immuno-electron microscopy reveals localization of c-Cbl and EGFR outside, and at clathrin-coated pits. COS-7 cells transfected with c-cbl cDNA were stimulated with EGF for 2 minutes at 37°C (iA,iB), for 60 minutes at 0°C (iC,iD,ii) or for 5 minutes at 37°C, followed by treatment for 15 minutes with saponin on ice (iii). All preparations were fixed and ultrathin cryosections were prepared for immunogold labelling. Sections were single labelled with antibodies to EGFR (iA,iC) or c-Cbl (iB,iD,iii), or double labelled with antibodies to EGFR and c-Cbl (iiA) or antibodies to clathrin and c-Cbl (iiB). Abbreviations: c-Cbl¹⁰, c-Cbl 10 nm gold particle; Clt¹⁵, clathrin 15 nm gold particle; EGFR¹⁵, EGFR 15 nm gold particle; N, the nucleus; S, cell surface. Inset in iii is a higher magnification of the marked area (asterisk) showing c-Cbl on coated vesicles. Magnification: i and ii show the same magnification. Bar, 100 nm.