Figure 4.

Dyngo-4a increases the amount of TMEM175 protein in the plasma membrane. (A). Schematic transmembrane topology of the HA₂-TMEM175 construct with the intracellular, N-terminal double influenza haemagglutinin (HA) tag. (B). The currents of the construct introduced in panel A are increased by the treatment of the oocytes with dyngo-4a (10 μM, 20 h, *** p < 0.005 compared to DMSO, Mann–Whitney U-test). The currents were measured in 80 mM K⁺ at −100 mV. (C). Anti-HA immunoblot of the crude plasma membrane preparations from the oocytes expressing HA₂-TMEM175 are shown. Eight groups of cells (n = 19 oocytes in each group) were treated with dyngo-4a (10 μM, Dyngo) or vehicle (DMSO), and in six groups, the proteins on the cell surface were digested with proteinase K (protK), as indicated below the graph. The proteolytic fragments are not visible on the immunoblot. No signal was detected in the control non-injected cells (non-inj., n = 19). Densitometry analysis of the bands is shown below the immunoblot. Note the lower intensity of bands in the Dyngo than in the DMSO groups. (D). Statistical analysis of the decrease of the anti-HA signals by proteinase K in the Dyngo and DMSO groups. * p < 0.05, (Student’s t-test, unpaired, homoscedastic) (E). TMEM175 structure was extended N-terminally with the single transmembrane segment of human CD8 protein, and an extracellular double-HA-tag was appended at the N-terminus. (F). The currents of the HA₂-CD8-TMEM175 construct introduced in panel E are increased by the treatment of the oocytes with dyngo-4a (10 μM, 20 h, *** p < 0.005 compared to DMSO, Mann–Whitney U-test). The currents were measured in 80 mM K⁺ at −100 mV. (G). Luminometry data (given in relative light units, RLU) of the oocytes expressing HA₂-CD8-TMEM175, and treated with dyngo-4a (10 μM, Dyngo) or vehicle (DMSO), as indicated below the graph. The signals were obtained by anti-HA indirect immunocytochemistry of fixed cells, followed by on cell surface horseradish peroxidase enhanced chemoluminescence (HRP-ECL) reaction. The background was determined by the identical reaction of non-injected oocytes. ** p < 0.02, (Student’s t-test, unpaired, homoscedastic) (H). TMEM175 was extracellularly HA-tagged by replacing a sequence of amino acids in the fifth EC loop with the HA epitope. (I). The currents of the loopHA-TMEM175 construct introduced in panel H shows the tendency to be increased by the treatment of the oocytes with dyngo-4a (10 μM, 20 h, p = 0.07 compared to DMSO, Mann–Whitney U-test). The currents were measured in 80 mM K⁺ at −100 mV. (J). Luminometry data with loopHA-TMEM175 in a similar experiment, as shown in panel G. * p < 0.05, (Student’s t-test, unpaired, heteroscedastic).