Figure 1

Characterization of TM2D3. (a) Expression of TM2D3 in transfected 293 T cells. 293 T cells were transiently transfected with expression vectors for indicated proteins or empty vector (Vector) as a control. Immunoblotting was performed with indicated antibodies. TM2D3v1 migrates slightly more slowly compared to TM2D3v2 probably due to its 26 additional amino acids. An anti-Vinculin blot serves as a loading control. (b) Induction of TM2D3 in Flp-In T-REx 293 cells by Tet. The cells stably transfected with vectors for indicated proteins were stimulated with Tet (1 μg/mL) for the indicated periods of time. Immunoblotting was performed with the indicated antibody. (c) Lower expression of TM2D3 in the stably transfected cells as compared to the transiently transfected cells. Flp-In T-REx 293 cells stably transfected with vectors for indicated proteins were stimulated with the indicated dosage of Tet for 20 h. Cell lysates used in a were loaded at the left-hand side as controls. Immunoblotting was performed with the indicated antibody. (d) Localization of TM2D3-FLAG (red) to vesicular structures in the cells and its slight overlap with the ER marker (green). Flp-In T-REx 293 cells stably transfected with either of the vectors for the indicated proteins or with their empty vector control (Vector) were transiently transfected with a vector for an ER fluorescent marker protein. At 1 day later, cells were stimulated with Tet (1 μg/mL) for 20 h and then fixed. The cells were stained with the indicated antibody and DAPI, and then confocal imaging was performed. The dotted boxes are enlarged in the top right panels. (e) Absence of colocalization of TM2D3-FLAG (red) and a Golgi marker (green). This experiment was performed as described in (d), except that the cells were transiently transfected with a vector for a fluorescent marker protein for the Golgi.