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).