Qae sephadex

Constructs encoding the WT and TMD mutant precursors were expressed in HEK 293 cells by transfection with the jetOPTIMUS transfection reagent from Polyplus (Illkirch, France) according to the manufacturer’s protocol. Cells in 6-well plates were harvested 48 h post-transfection and lysed in 100 μl of buffer A (25 mM Tris-Cl, pH 7.2, 150 mM NaCl, 1% Triton-X 100 and protease inhibitor cocktail). 30 μg of each cell lysate was incubated for 2 h at 37 °C in a buffer containing 100mM α-methyl mannoside, 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 10 mM MnCl₂, 75 μM UDP-[³H]GlcNAc (1 μCi), and 2 mg/mL bovine serum albumin in a final volume of 50 μl. The reactions were terminated by addition of 950 μl of 5 mM EDTA, pH 8.0. The sample was applied to a 1 mL column of QAE-Sephadex (GE Healthcare, Chicago, IL) equilibrated with 2 mM Tris base, pH 8.0. The column was washed with 5 mL of 2 mM Tris base and the phosphorylated products were eluted with 5 mL of 2 mM Tris base containing 30 mM NaCl. The incorporated [³H]GlcNAc-P was determined by addition of 8.5 mL of EcoLite scintillation fluid (MP Biomedicals Inc., Irvine, CA). The background activity in non-transfected cells less than 1% of that obtained with cells transfected with WT GlcNAc-1-phosphotransferase cDNA.

Constructs encoding WT and mutant proteins were expressed in GNPTAB^−/− HeLa cells or HEK 293 cells by transfection with jetOPTIMUS transfection reagent from Polyplus (Illkirch, France) according to the manufacturer’s protocol. Cells in 6-well plates were harvested 24 h post-transfection and lysed in 250 μl of buffer A (25 mM Tris-Cl, pH 7.2, 150 mM NaCl, 1% Triton-X 100 and protease inhibitor cocktail). 50 μg of cell lysate (5 μg in the case of cells expressing ΔS1-S3) in a final volume of 50 μl was incubated for 1 h at 37°C in buffer containing 100 mM α-methyl mannoside, 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 2 mM ATP, 75 μM UDP-[³H]GlcNAc (1 μCi), and 2 mg/mL bovine serum. The reactions were terminated by addition of 950 μl of 2 mM EDTA, pH 8.0. The sample was applied to a 1 mL column of QAE-Sephadex (GE Healthcare, Chicago, IL) equilibrated with 2 mM Tris base, pH 8.0. The column was washed with 5 mL of 2 mM Tris base and phosphorylated products were eluted with 5 mL of 2 mM Tris base containing 30 mM NaCl. The incorporated [³H]GlcNAc-P was determined by addition of 8.5 mL of EcoLite scintillation fluid (MP Biomedicals Inc., Irvine, CA). The background activity in non-transfected cells was less than 1% of that obtained with cells transfected with WT human GNPTAB cDNA.