Figure 2. TMEM87a/Elkin1 in WM266-4 cells.

(A) Transfection of WM266-4 cells with a plasmid encoding miRNA targeting Elkin1 leads to a reduction in Elkin1 transcript (Mann-Whitney, control n = 8, KD n = 6, *p=0.013). (B) Knockdown of Elkin1 leads to a significant decrease in MA currents in WM266-4 cells, in comparison with controls (two-way ANOVA, control n = 15, Elkin1 KD = 10, **p=0.004; Sidak’s multiple comparison, *p=0.02) (data presented as mean ± s.e.m.). (C) Transmembrane topology prediction of hsElkin1-iso1, with 6 TM domains (highest probability prediction of TM domains). (D) Western-blot analysis of samples prepared from HEK-293T cells overexpressing hsElkin1-isoform1 or hsElkin1-isoform3. The surface fraction was isolated by pull-down of biotinylated proteins after surface labelling. Note, both hsElkin1-isoform1 and hsElkin1-isoform3 are present at the cell surface. See Figure 2—figure supplement 1 for full blot. TIRF images of (E) hsElkin1-iso1-GFP, (F) Lifeact mCherry and (G) overlay in WM266-4 cells. Note that hsElkin1-iso1 is present in foci as well as the plasma membrane. TIRF images of (H) hsElkin1-iso3-GFP, (I) Lifeact mCherry and (J) overlay in WM266-4 cells. Note that hsElkin1-iso3 is present in the plasma membrane and associated with actin structures. Scale bar 10 µm. See Figure 2—videos 1 and 2 for corresponding live-cell imaging and Figure 2—figure supplement 2 for laser scanning confocal imaging of hsElkin1-iso1/hsElkin1-iso3 with a Golgi-RFP marker.

Figure 2—figure supplement 1. Cell-surface biotinlyation of hsElkin1-GFP fusion proteins.

hsElkin1-iso1-GFP or hsElkin1-iso3-GFP were overexpressed in HEK-293T P1KO cells. Cells were biotinylated and the cell-surface fraction isolated by pulldown of biotinylated proteins using Neutravidin beads. Input samples, the surface fraction and the supernatant were separated using SDS-PAGE, transferred to a PVDF membrane and Elkin-1-GFP fusion proteins were detected using an anti-GFP antibody. Both hsElkin1-iso1 and hsElkin1-iso3 were present in the plasma membrane fraction, as well as the supernatant.

Figure 2—figure supplement 2. Visualisation of hsElkin1-iso1 and -iso3 with laser-scanning confocal microscopy.

WM266-4 cells were transfected with plasmids encoding hsElkin1-iso1-GFP (A) or hsElkin1-iso3-GFP (D). Cells were additionally transduced with a vector encoding a Golgi-RFP marker (B,E). Overlays are presented in (C,F). It is clear that hsElkin1-iso1 localises partially, but not exclusively, to the Golgi. Scale bar, 10 µm.

Figure 2—video 1. hsElkin1-iso1-GFP dynamics in WM266-4 cells, as visualised with TIRF microscopy.

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Time-lapse TIRF images of WM266-4 cells expressing hsElkin1-iso1-GFP (green) and lifeact-mCherry (magenta). Scale bar 10 μm.

Figure 2—video 2. hsElkin1-iso3-GFP dynamics in WM266-4 cells, as visualised with TIRF microscopy.

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Time-lapse TIRF images of WM266-4 cells expressing hsElkin1-iso3-GFP (green) and lifeact-mCherry (magenta). Scale bar 10 μm.