Figure 4: Intracellular localisation of TMEM176A and TMEM176B.

(a) Human Th17 polarised cells from naive CD4⁺ T cells were coated on microscopy slides, fixed, permeabilised and co-stained for TMEM176B (red) and the indicated markers (green). DAPI was used for nuclear staining (blue). Arrows indicate TMEM176B colocalisation with the cis-Golgi protein GM130. Bar, 10 μm. (b) Pearson’s correlation coefficients of TMEM176B and the indicated markers (n = 10–15 in each group). (c) HeLa cells were treated or not with nocodazole for 4 hr and subsequently fixed, permeabilised and co-stained for TMEM176B (red), GM130 (cis-Golgi, green) and TGN46 (trans-Golgi and trans-Golgi network (TGN), blue). Bar, 10 μm. Insets represent higher magnifications of regions of interest. Linescans show fluorescence intensity along the lines overlaying the images. (d) HeLa cells were co-transfected with plasmids encoding TMEM176A-HA and TMEM176B-V5 fusion proteins and subsequently fixed, permeabilised and co-stained with HA (green) and V5 (red) monoclonal antibodies. DAPI was used for nuclear staining (blue). (e) Human Th17 polarised cells (as in A) were co-stained for GM130 (green), TMEM176A (purple) and TMEM176B (red). DAPI was used for nuclear staining (blue). Bar, 10 μm.