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. 2003 Sep 15;162(6):971–979. doi: 10.1083/jcb.200303018

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Copyright © 2003, The Rockefeller University Press

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Figure 3. — PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 5 μm.

Figure 3. — PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 5 μm.