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. 2010 Jun 22;18(8):1519–1526. doi: 10.1038/mt.2010.113

Figure 2.

Figure 2

Glycosylation analysis of NgHexA with an additional N-linked glycan containing mannose-6-phosphate residues in the α-subunit. Glycosylation of the α-subunit and the M6P content of HexA were examined by western blotting. (a) HexA fractions separated from CHO/HEXA/HEXB and CHO/NgHEXA/HEXB cell extracts obtained with the Vivapure spin column were treated with (+) or without (−) PNGase F. The α-subunit molecular masses were determined by western blotting using antihuman Hex α-subunit peptide antibodies. (b) HexA fractions separated from supernatants of the CHO/HEXA/HEXB and CHO/NgHEXA/HEXB cell lines with a DEAE column were analyzed by native-polyacrylamide gel electrophoresis (PAGE) and western blotting using concanavalin A. (c) The M6P contents of N-glycans of WT- and NgHexA, each exhibiting 4-methylumbelliferyl N-acetyl-β--glucosaminide-degrading activity (2,000 and 4,000 nmol/hour/lane), were determined by native-PAGE and lectin blotting using Dom9His.