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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Acta Neuropathol. 2018 Oct 31;137(1):71–88. doi: 10.1007/s00401-018-1925-9

Fig. 4. Biochemical characterization of rare variants in MFSD8 associated with FTLD risk.

Fig. 4.

(A-B) Five variants (S217G, E336Q, F379S, G385R, I492T) were generated, dual-tagged with GFP and HA (GFP-HA-MFSD8), transiently expressed in HEK-293T cells and compared to cells expressing human reference sequence (WT) MFSD8 and negative control, GFP-transfected cells. One day after transfection, the cells were lysed and the lysates analyzed by immunoblotting for MFSD8 using an HA antibody. The tagged MFSD8 constructs ran as a collection of specific bands ranging in size from approximately 55-85 kD. All variants displayed grossly normal overall expression, but one variant, F379S, frequently showed subtle increases in several distinct lower-molecular weight (MW) bands, relative to WT MFSD8. Immunoblots from two representative experiments are shown in (A-B), and the accumulated lower-MW bands for variant F379S are indicated by (*). Transferrin receptor (TfR) was used as a loading control. (C-D) Cell-surface biotinylation analysis revealed that variant F379S showed a modest but statistically significant increase in surface expression, relative to WT MFSD8. Cell surface-associated F379S also showed accumulation of a lower-MW band (*, C). TfR was used as a loading control for the cell lysates and as a positive control for the surface labeling. Results were quantified from three independent experiments. Differences with WT are significant at p < 0.01 (**).