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Fig. 10 | Molecular Neurodegeneration

Fig. 10

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

Fig. 10

Expression of NHE6 ΔES enhances apoptosis in AP-1 cells. a Confocal microscopy of fixed AP-1 cells expressing 3FNHE6v1HA WT (upper panels) or ΔES (lower panels). NHE6 was labelled with a mouse monoclonal anti-HAm antibody and an Alexa Fluor488-conjugated goat anti-mouse secondary antibody. Actin filaments were labelled with rhodamine-phalloidin and the nuclei were stained with DAPI. b Transient expression (48 h) of GFP, NHE6v1GFP WT or ΔES in AP-1 cells. Representative immunoblot probed with a polyclonal anti-GFP antibody (α-GFPP). c Flow cytometry analysis of AP-1 cells transfected with GFP alone, NHE6GFP WT or ΔES. Forty-eight hours after transfection, cells were labeled with Annexin V-APC and propidium iodide (PI) and 104 GFP-positive cells were examined by flow cytometry for each transfectant. Annexin V- and PI- double negative cells represent viable cells. Cells taking up only PI (PI+) are indicative of dead cells; Annexin V+ positive cells indicate early apoptotic cells whereas Annexin V+ and PI+ double positive cells represent late apoptotic cells. Results are shown as mean ± S.E.M. of eight independent experiments. Significance was determined using a paired two-tailed Student t-test, **p < 0.01. d AP-1 cells transiently expressing GFP alone, NHE6GFP WT or ΔES were isolated by cell sorting and then assayed for caspase 3/7 activity as described in “Materials and Methods”. Data were normalized to values for GFP-expressing cells and displayed as mean ± S.E.M. of four independent experiments, each done in triplicate. Significance was determined using a paired two-tailed Student t-test, *p < 0.05

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