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. 2010 Dec 20;286(7):5311–5318. doi: 10.1074/jbc.M110.202382

FIGURE 1.

FIGURE 1.

The γ-subunit of GlcNAc-1-phosphotransferase is glycosylated at Asn-88 and Asn-115. A, extracts (75 μg) from γ-subunit-overexpressing COS-7 cells were incubated for 1 h in the presence (+) or absence (−) of PNGase F, endoglycosidase H (endoH), or sialidase. SDS-PAGE (12.5% acrylamide) was performed under reducing conditions, and Western blot analysis was against the human γ-subunit (1:250 dilution). B, extracts (75 μg) from COS-7 cells overexpressing the wild-type (WT; lanes 2 and 3) and mutant (N88Q, N115Q, and N88Q/N115Q (N88/115Q); lanes 4-9) γ-subunits were incubated for 1 h in the presence (+) or absence (−) of PNGase F. The proteins were analyzed by SDS-PAGE (10% acrylamide) under reducing conditions, and human γ-subunit Western blot analysis was performed. Non-transfected cell extracts were used as a control (lane 1). The positions of the molecular mass marker proteins in kilodaltons are indicated. Representative blots of three to four experiments are shown.