To monitor the expression level and subcellular localization, WT and MmSVCT1 mutants containing a C-terminal GFP followed by a Strep tag were transfected into HEK293T cells (ATCC, #CRL-3216). The cells were maintained in DMEM (Cytiva) with 10% fetal bovine serum (HyClone) and 100 units/ml penicillin plus 100 µg/ml streptomycin. For western blot, cells were harvested 48 h after transfection and solubilized in a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 1% DDM for 1 h at 4 °C. After centrifugation at 20,000 × g for 30 min, equal amounts of samples were resolved on an SDS-polyacrylamide gel and transblotted onto a PVDF membrane. The membrane was then probed with 1:2000 diluted mouse anti-StrepII-tag monoclonal antibody (ABclonal, AE066, Clone No. AMC0521) and 1:5000 diluted goat HRP-conjugated anti-mouse IgG (BBI Life Sciences, D110087).
Immunofluorescence imaging was performed following the published protocol56 (link). HEK293T cells were grown on coverslips and transfected with MmSVCT1 variants. After 24 h, the cells were fixed with 3.7% formaldehyde in PBS buffer. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) from Sigma. Images were collected via the FITC channel (for the GFP signal) or DAPI channel on a DeltaVision deconvolution microscope (GE Healthcare). Data were captured and processed using DeltaVision softWoRx software V6.5.2 (Applied Precision).
Immunofluorescence imaging was performed following the published protocol56 (link). HEK293T cells were grown on coverslips and transfected with MmSVCT1 variants. After 24 h, the cells were fixed with 3.7% formaldehyde in PBS buffer. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) from Sigma. Images were collected via the FITC channel (for the GFP signal) or DAPI channel on a DeltaVision deconvolution microscope (GE Healthcare). Data were captured and processed using DeltaVision softWoRx software V6.5.2 (Applied Precision).