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tra12168-sup-0001-FigureS1.jpgJPEG image725KFigure S1: Activity of FGFR4-PA-GFP (A) and Rab11 knockdown efficiency in H1299 cells (B). A) H1299 cells transiently transfected with FGFR4 or FGFR4-PA-GFP were starved for 4 h and then stimulated for 15 min with 100 ng/mL FGF1 in the presence of 20 U/mL heparin. The cells were then lysed and the cell lysates were analysed by SDS-PAGE and western blotting with indicated antibodies. One representative experiment is shown. B) H1299 cells were transfected with siRNA oligos targeting Rab11a and b as indicated or a non-targeting siRNA control (scr) as described in Materials and Methods. The level of knockdown was assessed by western blotting. One representative experiment is shown.
tra12168-sup-0002-FigureS2.jpgJPEG image283KFigure S2: Binding of FGF1 to cells depleted for Rab11. U2OS cells stably expressing FGFR4 were transfected with non-targeting (scr) or Rab11-targeting siRNA oligos. Forty-eight hours after transfection, the cells were starved for 1 h and then kept at 4°C with 100 ng/mL FGF1 in the presence of 50 U/mL heparin. Next, the cells were washed and immediately lysed. Cell lysates were then analysed by SDS-PAGE and western blotting with indicated antibodies. One representative experiment is shown. FGF1 is indicated by the arrow and the asterisk indicates a background band.
tra12168-sup-0003-MovieS1.movapplication/unknown4939KMovie S1: H1299 cells transfected with photoactivatable FGFR4-PA-GFP were incubated with DL549-FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Transport of FGFR4 out from DL549-FGF1-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the DL549-FGF1-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 30 seconds over a period of 900 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0004-MovieS2.movapplication/unknown4374KMovie S2: H1299 cells transfected with a non-targeting siRNA control (scr) and photoactivatable FGFR4-PA-GFP were incubated with DL549-FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Transport of FGFR4 out from DL549-FGF1-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the DL549-FGF1-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0005-MovieS3.movapplication/unknown3818KMovie S3: H1299 cells transfected with siRNA oligos targeting Rab11a and photoactivatable FGFR4-PA-GFP were incubated with DL549-FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Transport of FGFR4 out from DL549-FGF1-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the DL549-FGF1-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0006-MovieS4.movapplication/unknown4453KMovie S4: H1299 cells transfected with siRNA oligos targeting Rab11b and photoactivatable FGFR4-PA-GFP were incubated with DL549-FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Transport of FGFR4 out from DL549-FGF1-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the DL549-FGF1-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0007-MovieS5.movapplication/unknown4885KMovie S5: H1299 cells transfected with siRNA oligos targeting Rab11a/b and photoactivatable FGFR4-PA-GFP were incubated with DL549-FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Transport of FGFR4 out from DL549-FGF1-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the DL549-FGF1-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0008-MovieS6.movapplication/unknown4752KMovie S6: H1299 cells co-transfected with photoactivatable FGFR4-PA-GFP and cherry-Rab11a were incubated with FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Recycling of FGFR4 from cherry-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the cherry-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0009-MovieS7.movapplication/unknown4811KMovie S7: H1299 cells co-transfected with photoactivatable FGFR4-PA-GFP and cherry-Rab11a S25N were incubated with FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Recycling of FGFR4 from cherry-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the cherry-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.
tra12168-sup-0010-MovieS8.movapplication/unknown4645KMovie S8: H1299 cells co-transfected with photoactivatable FGFR4-PA-GFP and cherry-Rab11-Fip2 were incubated with FGF1 (100 ng/mL) in the presence of heparin (50 U/mL). Recycling of FGFR4 from cherry-positive perinuclear vesicles was visualized using photoactivation, which was performed with a 405-nm laser aimed at the cherry-positive perinuclear vesicles (denoted by the green circle). Images were captured with a confocal microscope every 2 seconds over a period of 240 seconds. Movies were generated from these time-lapse images.

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