FIGURE 7.

The W207S mutant is degraded through the ubiquitin-proteasome pathway. A, HEK293T cells were transfected to express either EOGT^WT or EOGT^W207S. At 24 h after transfection, cells were treated with 20 μm MG132 for the indicated time. Cell lysates were subjected to immunoblotting with the indicated antibodies. B, HEK293T cells were transfected to express either FLAG-EOGT^WT (WT) or FLAG-EOGT^W207S (W207S). At 24 h after transfection, cells were treated with 20 μm MG132 for 90 min. Proteins from cell lysates were immunoprecipitated (IP) using an anti-FLAG antibody and immunoblotted as indicated. (C) HEK293T cells were cotransfected with N1EGF1–36-MycHis and EOGT constructs. After 42 h, the transfected cells were added to 20 μm MG132 for 6 h. Proteins from cell lysates were immunoprecipitated using anti-Myc antibody and subjected to immunoblotting with anti-O-GlcNAc (CTD110.6) or anti-Myc antibodies. D, HEK293T cells were transfected to express either FLAG-EOGT^WT (fWT) or FLAG-EOGT^Δ360–527 (fΔ360–527). At 24 h after transfection, cells were treated with 20 μm MG132 for 90 min. Cell lysates were immunoprecipitated using anti-FLAG antibody-conjugated beads and immunoblotted as indicated.