FIGURE 4.

Internalization of Met in response to LRR is clathrin- and Grb2-dependent. A, left panel, lysates from untransfected HeLa cells (Un) or cells transfected with control (Con) or CHC siRNA were examined by Western analysis using antibodies for CHC, transferrin receptor, Met, EEA1, and caveolin. Right panel, duplicate sets of control or CHC-depleted HeLa cells were co-treated for 10 min at 37 °C with Alexa⁵⁹⁴-Tfn and Alexa⁴⁸⁸-InlB, or Alexa⁴⁸⁸-LRR, and the relative amount of internalized ligand was analyzed by confocal microscopy. Values represent the mean fluorescence intensity ±S.E. from 2–3 experiments and are expressed as a percentage of control values. B, left panel, Western analysis was performed as indicated to confirm siRNA-mediated depletion of Grb2 in HeLa cells. Right Panel, the relative amounts of internalized Tfn, InlB, and LRR in a 10-min/37 °C pulse in HeLa cells transfected with control (Con) or Grb2 siRNAs were quantified by confocal microscopy. The average values of 2–3 experiments are expressed as a percentage of control values ± S.E. (*, p < 0.01, ANOVA).