Transfections, Immunocytochemistry and confocal microscopy
Human Osteosarcoma cells (Sigma-Aldrich, Darmstadt, Germany) were grown in DMEM (Sigma-Aldrich) complete medium to a confluency of 70 percent and then C-terminal GFP fused wild-type or mutant TMEM251 was transiently transfected using Lipofectamine 3000 (Thermo Fisher Scientific) following manufacturer’s protocol. Cells were fixed in 4% paraformaldehyde and blocked using 5% anti-goat serum in 0.1% triton X100/ PBS. Primary antibodies PDI mouse monoclonal antibody (Enzo Life Sciences, Nassau, NY) for Endoplasmic reticulum and Giantin mouse monoclonal antibody for Golgi complex (Enzo Life Sciences) were diluted in blocking medium to a final dilution of 1 in 100. Secondary staining was completed by using Goat anti-Mouse IgG Cross-Adsorbed Secondary Antibody (Thermo Fisher Scientific) diluted in blocking medium to a final dilution of 1 in 2000. Coverslips were mounted onto slides using Vectashield® hardset mounting media containing DAPI (Vector Laboratories, California, USA). Intracellular localization of both the wild-type and the mutant TMEM251 GFP fusions (GFP fused at C-terminal) were observed by confocal imaging of the transfected cells after immunostaining. Human Osteosarcoma cells were examined with LSM 700 Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany) equipped with Zeiss AxioCam camera (Carl Zeiss) and ZEN 2011 digital imaging software (Carl Zeiss).