Human embryonic kidney (HEK293T) cells expressing mutated glucose transporter 1-flag gene (MUT-GLUT1-flag) uptake less glucose than their WT-GLUT1-flag.

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Human embryonic kidney (HEK293T) cells expressing mutated glucose transporter 1-flag gene (MUT-GLUT1-flag) uptake less glucose than their WT-GLUT1-flag counterparts. Human embryonic kidney (HEK293T) cells expressing mutated glucose transporter 1-flag gene (MUT-GLUT1-flag) uptake less glucose than their WT-GLUT1-flag counterparts. (A) Evaluation of GLUT1 and GLUT1-flag protein expression in total cellular lysates of two independent sets of parental (non-transduced) HEK293T cells, cells transduced with empty vector, WT-GLUT1-flag and MUT-GLUT1-flag with Western blotting. β-actin levels served as loading control. (B) Absolute quantification of GLUT1-flag and β-2-microglobulin (B2M) transcripts in HEK-WT-GLUT1-flag and HEK-MUT-GLUT1-flag cells. (C) Relative quantification of GLUT1-flag transcript in HEK-WT-GLUT1-flag and HEK-MUT-GLUT1-flag cells. (D) [1,2-3H]-deoxy-D-glucose (2-DOG) uptake in two independent sets of HEK293T cells transduced with empty vector, WT-GLUT-flag, or MUT-GLUT1-flag. The figure presents mean counts per minute (cpm) value±SD; *p<0.05 in one-way analysis of variance (ANOVA) and Bonferroni's post hoc test. (E) 2-DOG uptake in the first set of HEK293T cells transduced with WT-GLUT-flag or MUT-GLUT1-flag incubated for 48 h with 1 μM lovastatin. The figure presents mean cpm as a percentage of WT-GLUT1-flag controls±SD; *p<0.05 in one-way ANOVA and Bonferroni's post hoc test. (F) Limited trypsin proteolysis of membrane proteins in HEK293T cells transduced with empty pLVX-IRES-Puro or WT-GLUT1-flag-encoding vector. The cells were preincubated for 48 h with 2.5 μM lovastatin. Each sample consisted of 1 mLn of cells. The cells were exposed to 0–10 μg/mL trypsin for 20 min. The figure presents result of Western blot using anti-GLUT1 antibody. Dominika Nowis et al. BMJ Open Diab Res Care 2014;2:e000017 ©2014 by American Diabetes Association