JCS032441 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Testing Rip11/FIP5 and RCP/FIP1 siRNAs. (A) Mock- or siRNA-transfected HeLa cells were incubated for 72 hours. Cells were then harvested and the levels of TfR, RCP/FIP1, Rip11/FIP5 and Kif3A/B tested with appropriate antibodies. Note that because anti-Kif3A/B antibodies recognize both Kif3A and Kif3B subunits of Kinesin II, Kif3B knockdown also results in downregulation of Kif3A. (B) Mock- or siRNA-transfected HeLa cells were incubated for 72 hours. Cells were then harvested and the levels of TfR, Rip11/FIP5 and Kif3A/B tested with appropriate antibodies. (C) Mock or Kif3B1 siRNA transfected HeLa cells were incubated for 72 hours. Cells were then harvested and the levels of KAP3A and Kif3A/B tested with appropriate antibodies. Note that although Kif3B knockdown also results in downregulation of Kif3A, it does not have any effect of the levels of KAP3A.

• Supplemental Figure S2 -

  Fig. S2. Measuring the levels of plasma membrane-associated transferrin receptors. (A,B) Plasma membrane-associated (non-permeabilized cells) or total cellular (saponin-permeabilized) TfR was measured by flow cytometry using either anti-TfR antibodies (A) or Alexa-Fluor-488-labeled Tf (Tf-Alexa488) (A,B). (C) HeLa cells were plated on collagen-coated glass coverslips and fixed with 4% paraformaldehyde. Cells were then incubated with 80 µg/ml Tf-Alexa488 (green) in the presence (permeabilized, bottom images) or absence (non-permeabilized, top images) of 0.4% saponin. Cells were then fixed again, washed, permeabilized with saponin and incubated with anti-TfR antibody conjugated to Texas Red (red). Blue is the nuclear stain. Scale bars: 5 µm. Yellow in merged image represents the overlap between Tf-Alexa488 and TfR.

• Supplemental Figure S3 -

  Fig. S3. Rip11/FIP5 and Kinesin II have no effect on lysosomal degradation of BSA and endocytic transport of EGFR. (A) The schematics of DQ-BSA-Green degradation assay. For more details see Materials and Methods. (B) Mock-treated cells or cells treated with RCP/FIP1 siRNA, Rip11/FIP5 siRNA1 or Kif3B siRNA1, were incubated with DQ-BSA-Green for 1 hour at 37°C. Cells were then washed and incubated for additional 2 hours at 37°C. The extent of DQ-BSA-Green degradation was then measured by flow cytometry. (C) Mock- or Rip11 siRNA1-treated HeLa cells were incubated for with Alexa-Fluor-594-labelled anti-EGFR antibody in the presence of 8 ng/ml EGF. Cells were then washed, acid-stripped and internalized anti-EGFR−Alexa-594 antibody was analyzed by flow cytometry. (D) Mock- or Rip11 siRNA1-treated HeLa cells were fixed and incubated with anti-EGFR−Alexa-594 antibody in the presence (total) or absence (plasma membrane) of saponin. Cells were then washed and the amount of cel-associated anti-EGFR−Alexa-594 antibody analyzed by flow cytometry.

• Supplemental Figure S4 -

  Fig. S4. Rip11/FIP5 colocalizes with microtubules. HeLa cells were fixed and stained with anti-Rip11 (A-C, green) and anti-tubulin (B and C, red) antibodies. Insets in A and C shown higher magnification images of the same cell. Rip11/FIP5-containing organelles are often arranged in the strings that follow microtubules. Scale bars: 5 µm (A), 2 µm (B), 2 µm (C).

• Supplemental Figure S5 -

  Fig. S5. Kif3B knockdown causes redistribution of Rip11/FIP5 to the tips of the cells. (A-C) HeLa cells were transduced with Kif3B siRNA1, fixed and stained with anti-Rip11/FIP5 (A and C, green) or anti-TfR (B and C, red) antibodies. Yellow in C represents the colocalization between Rip11 and TfR. Scale bar: 5 µm. (D,E) Mock (D) or Kif3B siRNA1-treated (E) HeLa cells were loaded for 30 minutes with Alexa-Fluor-488-labeled Tf. Cells were then washed and Tf-Alexa488 destaining analyzed by time-lapse microscopy in the presence of unlabeled Tf. Scale bar: 5 µm. (G) Mock- or Kif3B siRNA1-treated cells were loaded for 30 minutes with Tf-Alexa488. Cells were then washed and analyzed by time-lapse microscopy. Time-lapse, 15 minutes. Total of four images were taken. For bleaching control, four images of Tf-Alexa488-loaded cells were taken with a 1 second time lapse. Data shown are the means of the fluorescence intensity from four randomly chosen cells.

• Supplemental Figure S6 -

  Fig. S6. Kif3B is a putative Rip11/FIP5 binding protein. (A,B) Wild-type Rip11/FIP5 or Rip11/FIP5-Y629A binding to either Kif3B or KAP3 subunits of kinesin II was analyzed using yeast two-hybrid assay. Data shown are the mean ± s.d. of three independent experiments. (C) Rab11, Rab11-Q70L, or Rab11-S25N binding to either Kif3B or KAP3 were analyzed using yeast two-hybrid assay. Data shown are the mean ± s.d. of three independent experiments.

• Supplemental Figure S7 -

  Fig. S7. The effect of microtubule and actin depolymerization on Kif3B depletion-induced accumulation of the recycling endosomes at the periphery of the cell. Mock (A,B) or Kif3B siRNA-treated (C,D) HeLa cells were incubated for 30 minutes with 5 µg/ml Tf-Alexa488 in the presence (B,D) or absence (A,C) of nocodazole. Cells were then washed, fixed and analyzed by microscopy. Scale bars: 5 µm. (E-H) Mock (E,G,H) or Kif3B siRNA-treated (F) HeLa cells were incubated for 30 minutes with with 5 µg/ml Tf-Alexa488 in the presence (E,F,H) or absence (G) of 1 µM latrunculin A. Cells were then washed, fixed, stained with rhodamine-phalloidin (G,H) and analyzed by microscopy. Scale bars: 5 µm.