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. 2017 Jan 20;19(1):43–53. doi: 10.1016/j.neo.2016.11.007

Figure 5.

Figure 5

GALNT6 stabilizes GRP78 through O-glycosylation. (A) HeLa stable cells were treated with CHX (cycloheximide) prior to western blot analysis. Endogenous GRP78 was more stable in HeLa-GALNT6-WT stable cells when compared with -mock stable cells. (B) MDA-MB-435S-Mock and -GALNT6-WT stable cells were transfected with Flag-tagged GRP78 and treated with CHX prior to western blot. Overexpression of GALNT6 stabilized exogenously expressed GRP78 in MDA-MB-435S-GALNT6-WT stable cells. (C) We generated Flag-tagged constructs in which each of candidate O-glycosylation sites was substituted with an alanine residue (T85A, T151A, T166A, T184A, or T203A). (D) We established MCF7-derived cell lines that stably expressing each of Flag-tagged alanine-substituted GRP78 proteins, as well as Flag-tagged GRP78-WT protein. These stable cells were treated with CHX at different time points followed by western blot. Lower graph indicated Flag-tagged GRP78 protein intensities from western blot, which showed rapid reduction of GRP78 proteins harboring T151A, T166A, and T184A substitutions.