Fig. 5. OGT interacts with and stabilizes FAM134B.

a, b NP cells were cotransfected with HA-OGT and Flag-FAM134B. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag or anti-HA antibodies, and the precipitates were then immunoblotted with anti-Flag and anti-HA antibodies to show the interaction between HA-OGT and Flag-FAM134B. c, d NP cells were subjected to the indicated treatment, and the endogenous interaction between OGT and FAM134B was detected via coimmunoprecipitation. Then, the anti-OGT and anti-FAM134B immunoprecipitates were immunoblotted with anti-OGT and anti-FAM134B antibodies, respectively. e Flag-FAM134B-transfected NP cells were subjected to ND in the presence of TMG or OSMI-1 for 36 h. Cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with anti-Flag and anti-O-GlcNAc antibodies. f NP cells were subjected to treatment as indicated in e. Colocalization of endogenous FAM134B and O-GlcNAc was visualized by immunofluorescence staining. g After pretreatment with TMG or OSMI-1 for 24 h, NP cells were treated with CHX (50 µg/mL) for the indicated duration. The cells were harvested, and the FAM134B protein level was measured by western blot analysis. h NP cells were subjected to ND treatment in the presence of TMG and OSMI-1 for 36 h, and the proteasome inhibitor MG132 (10 µM) was added 6 h before cell collection. The protein level of FAM134B was assessed via western blot analysis. i Flag-FAM134B-transfected NP cells were subjected to ND in the presence of TMG or OSMI-1 for 36 h, and the proteasome inhibitor MG132 (10 µM) was added 6 h before cell collection. Anti-Flag immunoprecipitates of denatured proteins from cell lysates were then analyzed by western blotting with an anti-ubiquitin antibody. The data are presented as the mean ± SD values. ^∗∗P < 0.01, ^∗P < 0.05.