Figure S5.
Rab35 is required for the apical transport of sphingomyelin. (A) Wild-type EpH4 cells stably expressing Scarlet-Occludin and Rab35 KO cells were cultured together and stained with anti-Rab35 antibody (Green). Scale bar, 10 µm. (B) Total cell lysate of Rab35 KO cells was resolved by SDS-PAGE and immunoblotted with anti-Rab35 mAb and anti-alpha-tubulin mAb. (C) Subcellular localization of SS-GFP-Lys expressed in wild-type EpH4 cells and Rab35 KO cells. Scale bar, 10 µm. (D) Subcellular localization of GFP-tagged podocalyxin expressed in wild-type EpH4 cells and Rab35 KO cells. Scale bar, 10 µm. (E) EpH4 wild-type (WT) cells and Rab35 KO cells were treated with bSMase. bSMase was washed out and cells were cultured in normal medium for the indicated times. Recovery of sphingomyelin at the apical membrane was evaluated by staining with RFP-Lysenin. Scale bar, 20 μm. (F) Quantification of the number of RFP-Lysenin-positive cells at the apical membrane based on E. N ≥ 12 independent experiments; error bar, SD; *, P = 0.1008; ***, P = 0.0001; and ****, P < 0.0001 by Student’s t test. (G) Scanning electron microscopy of wild-type EpH4 cells and Rab35 KO cells. Scale bar, 5 µm. Source data are available for this figure: SourceData FS5.

Rab35 is required for the apical transport of sphingomyelin. (A) Wild-type EpH4 cells stably expressing Scarlet-Occludin and Rab35 KO cells were cultured together and stained with anti-Rab35 antibody (Green). Scale bar, 10 µm. (B) Total cell lysate of Rab35 KO cells was resolved by SDS-PAGE and immunoblotted with anti-Rab35 mAb and anti-alpha-tubulin mAb. (C) Subcellular localization of SS-GFP-Lys expressed in wild-type EpH4 cells and Rab35 KO cells. Scale bar, 10 µm. (D) Subcellular localization of GFP-tagged podocalyxin expressed in wild-type EpH4 cells and Rab35 KO cells. Scale bar, 10 µm. (E) EpH4 wild-type (WT) cells and Rab35 KO cells were treated with bSMase. bSMase was washed out and cells were cultured in normal medium for the indicated times. Recovery of sphingomyelin at the apical membrane was evaluated by staining with RFP-Lysenin. Scale bar, 20 μm. (F) Quantification of the number of RFP-Lysenin-positive cells at the apical membrane based on E. N ≥ 12 independent experiments; error bar, SD; *, P = 0.1008; ***, P = 0.0001; and ****, P < 0.0001 by Student’s t test. (G) Scanning electron microscopy of wild-type EpH4 cells and Rab35 KO cells. Scale bar, 5 µm. Source data are available for this figure: SourceData FS5.

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