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Fig. 7 | Molecular Neurodegeneration

Fig. 7

From: A Christianson syndrome-linked deletion mutation (∆287ES288) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

Fig. 7

Uptake of transferrin is impaired in HeLa cells expressing NHE6 ΔES. a Comparison of uptake of transferrin-Alexa Fluor633 (Tf-AF633; 10 μg/ml, 5 min) in AP-1 vs. HeLa cells. Significance was measured using a one-sample Student’s t-test; *p < 0.05. b Surface expression of transferrin receptor (TfR) in AP-1 vs. HeLa cells was measured by cell surface biotinylation. A representative immunoblot shows TfR and GAPDH expression in AP-1 and HeLa cells (left panel). Quantification of surface and total TfR relative to total GAPDH expression from three different experiments; values are normalized to the TfR/GAPDH ratio in AP-1 cells (right panel). c Transient expression of GFP, NHE6v1GFP WT or ΔES in HeLa cells after 48 h. Representative immunoblot probed with a polyclonal anti-GFP (α-GFPP) antibody. d Uptake of Tf-AF633 was monitored in HeLa cells expressing GFP, NHE6v1GFP WT or ΔES. Median fluorescence intensity (M.I.F.) of Tf-AF633 was measured in 104 GFP-positive cells by flow cytometry. Data were normalized and represent mean ± S.E.M. of eight different experiments. Significance was established using a one-way ANOVA followed by a Tukey test, **p < 0.01. e Uptake of Tf-AF633 in HeLa cells expressing GFP, NHE6v1GFP WT or ΔES in the absence (−) or presence (+) of 10-fold excess unlabeled Tf. The M.I.F. of Tf-AF633 was measured in 104 GFP-positive cells by flow cytometry. Data represent mean ± S.E.M. of three different experiments, **p < 0.01. f qPCR of NHE6 mRNA levels in HeLa cells treated for 72 h with non-target (scrambled) siRNA or NHE6 siRNA. g Uptake of Tf-AF633 was monitored in HeLa cells expressing non-target siRNA or NHE6 siRNA for 72 h. The M.I.F. of Tf-AF633 was measured in 105 cells by flow cytometry. Data were normalized and represent mean ± S.E.M. of three different experiments. Significance was established using a one-sample Student’s t-test, **p < 0.01. h Cell surface levels of TfR in HeLa cells transiently expressing GFP, NHE6v1GFP WT or ΔES were determined by cell surface biotinylation. Representative immunoblot of TfR expression at the cell surface and the total fraction is shown in the left panel. Quantification by densitometry of cell surface/total TfR levels from three different experiments is shown in the right panel. Values were normalized to TfR amounts present in GFP-expressing cells and represent the mean ± S.E.M. Significance was established using a one-sample Student t-test, ** p <0.01, * p <0.05

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