Journal:

Article Title: Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

doi: 10.1091/mbc.E04-01-0041

Figure Lengend Snippet: Recruitment of Grb2, Cbl, and Eps15 to the plasma membrane upon incubation with EGF on ice. (A) Hep2 cells, incubated without ligand (control) or with EGF (60 ng/ml) for 60 min on ice, were prepared for immunofluorescence and labeled for Grb2, Cbl, or Eps15, respectively. On incubation with EGF on ice, Grb2, Cbl, and Eps15 were recruited to the plasma membrane. Bar, 10 μm. (B) Thawed cryosections of serum-starved Hep2 cells, incubated with EGF (60 ng/ml) on ice for 60 min, were either single-labeled for Grb2 (I) or double-labeled for Grb2 (large gold particles) and either EGF (II) or clathrin (III, IV; small gold particles). Note that although coat-associated labeling for Grb2 (large arrows in I-IV) is restricted to the rim of the coated area, labeling for EGF (small arrows in II) is all along the coat. The double-labeling for Grb2 and clathrin confirmed that Grb2 is associated both with flat and invaginated clathrin-coated plasma membrane. Sections from the same specimen were either labeled only for Cbl (V–VI), or both for Cbl (large gold particles) and clathrin (small gold particles; VII and VIII). Note that the coat associated labeling for Cbl (arrows) is restricted to the rim of the coated area. The double-labeling for Cbl and clathrin confirmed that Cbl is associated with both flat and invaginated clathrin-coated plasma membrane. Bars, 100 nm.

Article Snippet: Rabbit anti-Eps15 (C-term) was from BAbCO (Richmond, CA), and rabbit anti-human transferrin receptor antibody from HybriDomus (Hellebaek, Denmark).

Techniques: Incubation, Immunofluorescence, Labeling