Figure 2.

Cells expressing GNE or GNE mutants produced sialic acid. Cytosolic (A), nuclear (B), and membrane-associated (C) sialic acid were quantified by DMB derivatization with detection by fluorescent HPLC. Cell surface sialic acid was measured by flow cytometry using MAL-II lectin to detect α2–3-linked sialic acid (D) and SNA lectin to detect α2–6-linked sialic acid (E). F, cell lines labeled in black produce sialic acid, whereas cell lines labeled in red did not express an active GNE epimerase domain and consequently cannot synthesize sialic acid. For all panels, cells were cultured for 24 h, and data shown represent three biological replicates, with error bars depicting the mean and S.E. Each data point represents the MFI of a single sample, typically of 10,000 cells. Flow cytometry experiments were performed at least twice. Statistical significance determined by unpaired Welch's test: ** indicates a p value < 0.01, and * indicates a p value < 0.05. ns indicates difference not statistically significant.