FIGURE 5.

Rab5 appears to act upstream of Rab31 in EGFR trafficking. A, A431 cells were transfected with either mCherry-Rab5 or mCherry-Rab31. Cells were pulsed with 0.5 μg/ml EGF-FITC and fixed at the various time points indicated for immunofluorescence analysis. The percentage of EGF-FITC positive puncta that were positive for either Rab5 or Rab31 was quantified from cells fixed after a 0-, 10-, 30-, and 60-min chase and presented graphically as a percentage of total EGF-FITC puncta counted. 29 cells in three independent experiments were analyzed, and data are shown as mean ± S.E. B, A431 cells were transfected with scrambled (Scr), Rab5, or Rab31 siRNA as indicated, and subsequent analyses were performed after 48 h. Top panel, the extent of knockdown was assessed by RT-PCR. Bottom panel, cells were pulsed with 0.5 μg/ml EGF-TxR and fixed at 30 min. Cells were immunostained for CD63, and the percentages of EGF-TxR puncta that were also positive for CD63 were quantified and presented graphically as a percentage of total EGF-TxR puncta counted. 27 cells in three independent experiments were analyzed, and data are shown as mean ± S.E.*, p < 0.05; **, p < 0.01; Student's t test.