Figure 2.

SNX27 is essential for maintaining ASCT2 levels at the plasma membrane. A, relative mRNA expression of SNAT1, SNAT2, and ASCT2 in HeLa control and SNX27 KO cells were determined by TaqMan gene expression assays and normalized to the expression of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase. The graph represents the difference of gene expression within HeLa control and SNX27 KO cells (means ± S.D.). A.U., arbitrary units. B, glutamine-starved HeLa and SNX27 KO HeLa cells were incubated with glutamine-free DMEM supplemented with 0.5 μCi/ml 2-deoxy-H³-glutamine for 15 min. Cells were harvested, and incorporated radioactivity was quantified on a MicroBeta liquid scintillation counter. The counts per minute (cpm) were normalized to total protein concentrations calculated by BCA assay, and the value presented represents the -fold-difference in glutamine uptake (means ± S.D.) from three experiments. Two-tailed Student's t test indicates the difference between HeLa and SNX27 KO HeLa cells. *, p < 0.05. C, HeLa and SNX27 KO HeLa cells were subjected to subcellular fractionation to generate purified PM, intracellular membrane, as well as cytosolic fractionations. Equal amounts of protein from whole-cell lysates (WCL, 30 μg) and each fraction (10 μg) were used for Western blotting to determine the protein expression of ASCT2, Na⁺/K⁺-ATPase, TfR, SNX27, and tubulin as indicated. Representative blots from at least three independent experiments are shown, and the calculated molecular weight for each protein is indicated. The -fold differences for ASCT2, TfR, and Na⁺/K⁺-ATPase between HeLa cells and SNX27 KO HeLa cells are presented (means ± S.D.). Two-tailed Student's t test indicates the difference between HeLa and SNX27 KO HeLa cells. *, p < 0.05, HeLa versus SNX27 KO cells.