Figure 9.

Endocytosis of EGF and EGFR is inhibited upon overexpression of ubiquitin that cannot be conjugated to endogenous proteins. Hep2 cells on coverslips were transfected with Myc-tagged UbR (A–C) or with Myc-tagged UbR L8A/I44A (D) for 48 h. Rhodamine-EGF (10 ng/ml, A and D), EGF (100 ng/ml, B), or Alexa594-Tf (50 μg/ml, C) was added to the cells at 37°C for 15 min. The cells were fixed and immunostained using mouse anti-Myc antibodies and secondary Rhodamine Red-X– or FITC-conjugated antibodies to detect transfected cells (left columns). Cells in B were additionally immunostained with anti-EGFR antibodies, followed by secondary FITC-conjugated antibodies. Bar, 5 μm.