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. 2019 Mar 21;14(3):e0214059. doi: 10.1371/journal.pone.0214059

Fig 3. Tax-1 Expression altered the Cellular Localization of GLUT1.

Fig 3

A) Tax-1 over-expression had no effect on SNX27 steady state levels. HEK293T cells were transfected with a titration of S-tag Tax-1 plasmid (100 ng to 2,000 ng). 24 hours post transfection cells were collected and lysed. Samples were measured for protein concentration and 10 μg of protein were from each sample were subjected to SDS-PAGE followed by immunoblotting. The membrane was probed for SNX27, S-tag, and Actin as shown. B and C) Tax-1 over-expression lowered the amount of GLUT1 located on the surface of cells. HEK293T cell were transfected with empty vector, S-tag Tax-1, or S-tag Tax-1 ΔPBM (B) or siRNA targeting SNX27 or a scramble control (C). 24 hours post transfection cells were collected and stained with the GLUT1-RBD-GFP ligand per manufacturer’s instructions. Cells were then measured for GFP expression via flow cytometry. The histograms show the transfected cell populations with GFP intensity on the X-axis and number of cells on the Y-axis. A portion of the samples were lysed and subjected to SDS-PAGE followed by immunoblotting with the antibodies for SNX27, S-tag, or Actin, as indicated. D) Tax-1 over-expression increases colocalization of GLUT1 with LAMP1. Coverslips were seeded with HeLa cells. Cells were transfected with either empty plasmid, S-tag Tax-1, scramble siRNA, or siRNA against SNX27. 24 hours post transfection cells were fixed, permeabilized, and probed overnight with antibodies against GLUT1 and LAMP1. Alexaflour secondary antibodies were used. Coverslips were mounted on slides and deconvolution microscopy was performed using a DeltaVision microscope. Red represents GLUT1 staining, green represents LAMP1 staining, and blue represents DAPI staining. E) Calculated Pearson's coefficient of correlation for all conditions. Statistical analysis was performed using a one-way analysis of variance with Dunnett’s multiple comparison test. ** = p < 0.005, * = p <0.05. F) Western blot analysis of cells used in immunofluorescence assays. SNX27, S-tag, and Actin antibodies were used as indicated.